Apparatus and method for dispensing fluid, semi-solid and solid samples

ABSTRACT

The invention relates generally to the field of automated collection and deposition of fluid, semi-solid, and solid samples of biological or chemical materials. More specifically, the invention relates to the field of microarrayers, which are devices for autonomously depositing minute droplets of biological or chemical fluid samples in ordered arrays onto substrates. The invention also relates to tissue arrayers, which are devices for the collection and deposition of solid and semi-solid tissue samples in ordered arrays. Other aspects of the invention relate to fluidics robots, which are devices for the autonomous collection, dispensing and processing of biological or chemical fluid samples. The invention improves the throughput of microarrayers, tissue arrayers, and fluidics robots by providing methods and apparatuses to precisely and repeatably load supplies into the machines.

CROSS REFERENCE TO RELATED APPLICATIONS

This application incorporates by reference in its entirety and claimspriority to U.S. Provisional Patent Application Ser. No. 60/514,285,entitled “Microarrayer,” filed on Oct. 24, 2003.

FIELD OF THE INVENTION

The present invention relates generally to the field of automatedcollection and deposition of fluid, semi-solid, and solid samples ofbiological or chemical materials, for example, using a microarrayer.

BACKGROUND

The ability to produce arrays of fluid or tissue samples is of greatvalue for increasing the rate at which chemical or biological studiesmay be performed, and the use of such arrays has been widely adopted inthe genomics research, biological research and drug-discoveryindustries.

Microarrayers are automated instruments used to deposit or spot minuteamounts of chemical or biological substances, such as DNA, RNA, cDNA,polynucleotides, oligonucleotides, and proteins in a dense array ofminute fluid droplets on a substrate, such as a glass slide. The generalpurpose of fabricating microarrays is to permit massively parallelinvestigation of chemical or biological activity. The microarray formatallows hundreds, thousands, tens of thousands or hundreds of thousandsof assays to be performed in parallel, enabling experiments andinvestigations that would have previously taken years, to be performedin a matter of days.

Therefore, the ability to produce spotted microarrays in large quantity,rapidly, at reasonable cost, and with uniform and consistent depositionproperties, such as spot size, shape and density, has significantindustrial and economic importance.

SUMMARY

Several microarray spotting techniques have been developed in recentyears to automatically deposit droplets of chemical and biologicalsubstances, in a liquid state, onto solid substrates. As used in thisdisclosure, the term “drop” or “droplet” refers to a very small quantityof fluid, and not to any particular shape of the fluid volume. Thedeposit elements used to spot a fluid on a substrate includes ink-jets,pens, quill pins, and solid pins. In each spotting technique, adeposition element acquires fluid from a fluid reservoir and spots thedroplets in the desired position on the substrate.

The simplest, and perhaps most robust printing method uses solid pins.Significant advantages of solid pins are their simplicity andreliability, ease of cleaning and their relative lack of sensitivity tothe sample fluid viscosity. An additional benefit of the use of solidpins is minimal sample fluid wastage. Since only a single droplet iscaptured by the pin, little or no sample fluid is lost at the cessationof printing with that sample. Disadvantages of spotting with solid pinsinclude variations in spot size and intensity resulting from differencesin the evaporation of the fluids being carried by the pins. We havedetermined that these variations result from the different times ofexposure to the air of the fluid droplets for different depositionpaths. The exposure time differences are significant considering thefluid volumes carried by such solid deposition pins are in the picoliterto nanoliter range. Therefore, a need exists to eliminate thesevariations, for example, by providing equal exposure time to fluiddroplets held by the deposit element as the deposit element travels fromthe well plate to the substrate. Another disadvantage of existingsolid-pin microarrayers is their lower spot deposition rates compared toquill pins, pens and ink-jets because of the requirement for the soliddeposition pin to reacquire fluid from a fluid reservoir after everydeposition. A need exists, therefore, for increasing the deposition rateof solid-pin microarrayers.

Existing microarrayers have used several motion architectures (the term“architecture” being used herein to describe the general design of theassembly and its fluid capture and fluid-droplet deposition operations).

Current microarrayer architectures are often inefficient, lackflexibility, have limited throughput, and/or produce microarrays thathave a lack of uniformity in the deposition of the fluid droplets. Forexample, we have determined that variability in the thickness of thesubstrates which are loaded into the system creates uncertainty in theheight of the surface upon which the fluid droplets are deposited andcauses undesirable variations in droplet deposition from substrate tosubstrate. The uncertainty in the height of the fluid deposition surfaceis of particular concern for non-contact printing with quill pins, solidpins, and pens, since it is desired to accurately touch only thedroplets of fluid on the tip of the device upon the substrate, and notthe tip itself, to prevent potential damage to delicate substratesurfaces. Traditional microarrayers do not include means forcompensating for the lack of uniformity in substrate thicknesses.

Another disadvantage of current microarrayers is that the substrates andwell plates need to be manually positioned inside the microarrayer. Inaddition to being time consuming, which decreases throughput, thisprocess leads to errors resulting from frequent human access to thedeposition area. Moreover, traditional microarrayers do not includemeans for loading the substrates and the well plates into the system inan accurate, repeatable manner. We have determined that his hinders theaccurate deposition of fluid samples on the substrates.

Therefore, a need exists to improve the apparatuses and procedures usedfor loading substrates into, and unloading substrates from, amicroarrayer. A need also exists to provide a microarrayer that canaccommodate substrates having variable thicknesses while minimizingundesirable variations in droplet deposition from substrate tosubstrate.

In another aspect, the invention also relates to the more general fieldof dispensing samples in array formats. Tissue arrays, which are arraysof thin slices of tissue cores, are typically formed in a multi-stepprocess. Typically a piece of biological tissue is formalin-fixed andembedded in a paraffin block, known as the donor block. Small cores ofthe semi-solid paraffin-embedded tissue (typically about 0.5 millimetersto a few millimeters in diameter) are then removed from the donor blockwith a tubular cutting device and deposited in an ordered array withinmatching, vertically-oriented cylindrical recesses in a receivingparaffin block. The receiving paraffin blocks are then thinly sliced inthe horizontal plane and the slices are transferred to supportingsubstrates. The slices of the receiving paraffin block forming thearrays are typically less than 10 microns thick. The receiving paraffinblock can therefore produce many copies of the array of core samples,which is of great value and importance for parallel biologicalexperimentation. Tens, hundreds, or thousands of tissue samples can beplaced on a tissue array. The solid cores from frozen tissue can bedeposited in a similar manner to that described for formalin-fixedsemi-solid tissue arrays.

Currently, tissue arrays are most typically produced by manual means,aided in some instances by un-powered, passive, mechanical stages toalign the elements of the array deposited into the receiving paraffinblock. Many hours and much manual labor are required to produce a tissuearray of a few hundred elements. The present limitations in flexibility,speed, and accuracy are significant impediments to the adoption of thisimportant technology.

A further limitation of current tissue arrayers is that paraffin-blockmounting arrangements have been bottom-referenced, i.e. the block ismounted such that its bottom surface rests upon a reference plane;however, core-formation and core deposition occurs at the top surface.Paraffin blocks are not typically cast to precise dimensionaltolerances. Since they are bottom referenced in current systems,uncertainty and variability exists in the location of the top surface ofthe block where tissue cores are deposited. We have determined that thiscan lead to inconsistent and inaccurate removal of tissue cores fromdonor blocks and inconsistent and inaccurate core-placement in receiverblocks.

Therefore, a need exists to improve the apparatuses and procedures usedfor loading and unloading donor blocks and receiver blocks into and outof a tissue arrayer. A need also exists to provide a tissue arrayer thatcan accommodate donor blocks and receiver blocks that vary in height toachieve more consistent and accurate removal of tissue cores from donorblocks and more consistent and accurate core-placement in receiverblocks. A need also exists to improve the speed of creating tissuearrays using an automated process.

A further dispensing application covered by the present inventionrelates to the field of fluidics handling systems, the devices thatperform these tasks commonly known as fluidics robots. Generally,fluidics handling systems are used to transfer fluids between a fluidsource reservoir and a fluid target reservoir. In addition, assays canbe automatically prepared and processed, including, in some instances,operations such as mixing, filtering, heating and cooling. In someprior-art applications, centrifugation and polymerase chain reactionsteps are also provided. Fluidics handling systems play a significantrole within the life science industry for automating fluid dispensing,fluid transfers, assay preparation, and assay processing.

One disadvantage of prior art fluidic robots is that reconfiguration ofexisting machines to accommodate different numbers, sizes, or styles ofreservoirs or other elements requires manual re-configuration of themounting provisions on a fixed platen. This results in limitedflexibility of existing designs and significant time lost to the manualre-configuration. Therefore, a need exists to improve the apparatusesand methods for reconfiguring a fluidic robot to handle differentnumbers, sizes, or styles of source reservoirs and target reservoirs.

In one aspect the invention relates to a method of depositing at leasttwo minute droplets of fluid on a substrate. The method includes thesteps of supplying a first fluid to a deposit element by dipping thedeposit element into the first fluid in a fluid reservoir, moving atleast one of the deposit element and the substrate relatively to deposita droplet of the first fluid at a first location on the substrate,supplying a second fluid to the deposit element by dipping the depositelement into the second fluid in the fluid reservoir and moving at leastone of the deposit element and the substrate relatively to deposit adroplet of the second fluid at a second location on the substrate. Avolume of the first fluid carried by the deposit element and a volume ofthe second fluid carried by the deposit element are exposed to asurrounding atmosphere for substantially a same amount of time betweentheir respective extractions from the fluid reservoir and theirrespective depositions on the substrate by controlling at least one ofspeed and timing of relative motion between the deposit element and thesubstrate. In one embodiment, the first fluid and the second fluid areobtained from a substantially same location in the fluid reservoir.

In another aspect, the invention relates to a method of repeatedlydepositing minute droplets of fluid on a substrate. The method includescapturing fluid on a deposit element and moving at least one of thedeposit element and a substrate relatively to deposit the fluid on thesubstrate such that each deposition occurs at a same determinable timeafter capturing the fluid on the deposit element. The time isdeterminable by at least one of adjusting relative-motion velocity ofthe deposit element and the substrate and introducing a motion delay toone of the deposit element and the substrate.

In another aspect, the invention relates to a tissue arrayer. The tissuearrayer includes a coring head for extracting a core sample from a donorblock and depositing the core sample in a receiving block. The tissuearrayer also includes a removable block-holder for holding at least oneof the donor block and the receiving block, the removable block-holderincluding an apparatus for precisely and repeatably positioning theremovable block-holder on a block-holder support.

In one embodiment, the block-holder support includes a first datum forengaging the removable block-holder and for restricting movement of theremovable block-holder along an x-axis and a mutually orthogonal y-axis,and defining a first point in a z-axis, the z-axis mutually orthogonalto the x-axis and the y-axis, a second datum for engaging the removableblock-holder and for at least partially locating the removableblock-holder along at least one of the x-axis and the y-axis anddefining a second point in the z-axis, and a third datum for engagingthe removable block-holder and defining a third point in the z-axis. Inone embodiment, the first datum includes at least a portion of a spherefor engaging a conical recess formed in the removable block-holder. Inanother embodiment, the second datum is engageable with a linear recessformed in the removable block-holder.

In another embodiment in accordance with the invention, the apparatusfor precisely and repeatably positioning the removable block-holder onthe block-holder support includes a first element for mating with afirst datum, and for restricting movement of the removable block-holderalong an x-axis and a mutually orthogonal y-axis and defining a firstpoint in a z-axis, the z-axis mutually orthogonal to the x-axis and they-axis. The apparatus also includes a second element for mating with asecond datum, and for at least partially locating the removableblock-holder along at least one of the x-axis and the y-axis whiledefining a second point in the z-axis and a third element for matingwith a third datum and defining a third point in the z-axis. The firstelement may include a conical recess for mating with the first datum. Inanother embodiment, at least two of the elements are adjustable alongthe z-axis.

In another embodiment, the removable block-holder includes a top surfaceand a bottom surface spaced from the top surface, an aperture extendingat least partially between the top surface and the bottom surface, anintersection of the aperture and the top surface defining a perimeter,and at least three reference points proximate the perimeter and defininga reference plane, the reference points for engaging a top surface of atleast one of the donor block and the receiving block when at least oneof the donor block and the receiving block is disposed in the aperture.In one embodiment, the removable block-holder includes referencesurfaces for engaging and precisely locating a top surface of at leastone of the donor block and the receiving block in a known plane withrespect to the removable block-holder. In another embodiment, theremovable block-holder further includes a removable block-mountingfixture onto which at least one of the donor block and the receivingblock is mountable. The removable block-mounting fixture includes alocking element for securing the removable block-mounting fixture in theremovable block-holder and for biasing the top surface of at least oneof the donor block and the receiving block against the at least threereference points.

The tissue arrayer can also include a storage for storing at least oneremovable block-holder and means for transferring at least one removableblock-holder between the storage and the block-holder support. Inanother embodiment, the removable block-holder support includes a donorblock-holder support and a receiver block-holder support and the donorblock-holder support and the receiver-block holder support are eachconstrained to move within a plane substantially perpendicular to thecoring head when disposed beneath the coring head. In anotherembodiment, the plane of motion of the donor block-holder is displacedfrom a plane of motion of the receiver-block holder. In a furtheradaptation, the tissue arrayer includes a core filling head fordepositing material into a void created in the donor block by the coringhead.

In another embodiment, at least one of the donor block, the receiverblock, the donor block-holder support and the receiver block-holdersupport, include a tracking device. The tracking device includes atleast one of a barcode, a radio-frequency identification (RFID)transponder programmed with a unique code readable by an RFIDinterrogator by non-contact means, and a semi-conductor memory deviceprogrammed with a unique code. The semi-conductor memory device isreadable by at least one of an electric sensor, and an external sensorthat is in communication with the semi-conductor memory device throughat least one of optical, infra-red, and radio-frequency communication.In yet another embodiment, the tissue arrayer includes means for locallystoring and updating information on at least one of the donor block, thereceiver block, the donor block-holder support and the receiverblock-holder support. The means includes at least one of aradio-frequency identification (RFID) transponder that is dynamicallyprogrammable onto at least one of the donor block, the receiver block,the donor block-holder support and the receiver block-holder support,the transponder readable by an RFID interrogator by non-contact means,and a semi-conductor memory device. The semi-conductor memory device isdynamically programmable onto at least one of the donor block, thereceiver block, the donor block-holder support and the receiverblock-holder support. The semi-conductor memory device is also readableby at least one of electrical contact and an external sensor that is incommunication with the semi-conductor memory device through at least oneof optical, infra-red, and radio-frequency communication.

In another embodiment, overall removable block-holder and block-holdersupport system position accuracy is within ±0.02″ in x, y, and z-axes.In a preferred embodiment, overall removable block-holder andblock-holder support system position accuracy is within ±0.002″ in az-axis and within ±0.01″ in x and y axes. In a more preferredembodiment, overall removable block-holder and block-holder supportsystem position accuracy is within ±0.0002″ in a z-axis and within±0.001″ in x and y axes.

In another aspect, the invention relates to a method of extractingtissue core samples from a donor block and depositing the core samplesin a receiver block. The method includes the steps of providing a donorblock including a tissue sample to be cored and providing a coring headfor extracting a tissue core from the donor block. The method alsoincludes obtaining an image of a surface of the donor block to be coredby the coring head and selecting and recording positional information ofa coring location from the obtained image. The method also includesinitiating autonomous tissue core sampling at the coring location usingthe selected and recorded positional information.

In another aspect the invention relates to a receiver block containingthe extracted core tissue samples in accordance with the method justdescribed.

In one embodiment, the method includes the step of providing a receivingblock and depositing the extracted tissue core into the receiving block.The method can also include the step of filling a void created in thedonor block created by the tissue core sampling with a filling material.The step of obtaining an image of the surface of the donor block canfurther include providing a high resolution camera at a known positionfrom the coring head and providing a high resolution video display todisplay the image of the donor block. As a further step, the method mayinclude providing at least one positional reference in a field of viewof the camera for establishing an offset distance of the camera to thecoring head and to correct non-linearities in the displayed image of thedonor block.

In another embodiment, the method includes the step of mounting at leastone of the donor block and the receiving block on a removable holder,the removable holder including an apparatus for precisely and repeatablypositioning the removable holder on a block-holder support.

In another aspect, the invention relates to a fluidics handling systemfor transferring a fluid from a fluid-source reservoir to a fluid-targetreservoir. The fluidics handling system includes at least one dispensinghead and a removable holder for holding at least one of a removablefluid-source reservoir and a removable fluid target reservoir, theremovable holder including an apparatus for precisely and repeatablypositioning the removable holder on a holder-support.

In one embodiment, the holder-support includes a first datum forengaging the removable holder and for restricting movement of theremovable holder along an x-axis and a mutually orthogonal y-axis, anddefining a first point in a z-axis, the z-axis mutually orthogonal tothe x-axis and the y-axis. The holder-support also includes a seconddatum for engaging the removable holder and for at least partiallylocating the removable holder along at least one of the x-axis and they-axis and defining a second point in the z-axis. A third datum is alsoincluded in the holder-support for engaging the removable holder anddefining a third point in the z-axis. In one embodiment, the first datumincludes at least a portion of a sphere for engaging a conical recessformed in the removable holder. In another embodiment, the second datumis engageable with a linear recess formed in the removable holder.

In another embodiment, the apparatus for repeatably positioning theremovable holder on the holder-support further includes a first elementfor mating with a first datum, and for restricting movement of theremovable holder along an x-axis and a mutually orthogonal y-axis anddefining a first point in a z-axis, the z-axis mutually orthogonal tothe x-axis and the y-axis. The apparatus also includes a second elementfor mating with a second datum, and for at least partially locating theremovable holder along at least one of the x-axis and the y-axis whiledefining a second point in the z-axis. A third element is also includedin the apparatus for mating with a third datum and defining a thirdpoint in the z-axis. The first element may include a conical recess formating with the first datum. In one embodiment, at least two of thethree elements are adjustable along the z-axis.

The fluidics handling system may also include a storage for storingremovable holders. In addition, means for transferring the removableholder between the storage and the holder support may be included. Inone embodiment, the removable holder is moved from the storage to theholder-support by moving the removable holder in a vertical directionwithin the holder storage to dispose the removable holder onto theholder-support and moving the holder support in a horizontal directionto retract the holder support from the storage.

In another embodiment, the dispensing head includes a plurality ofdispensing elements, the dispensing elements moveable along at least oneof an x-axis and a mutually orthogonal y-axis relative to each other toalter the distance between tips of the dispensing elements. In yetanother embodiment, the dispensing head is constrained to move along asingle axis. The removable holder may be constrained to move within aplane perpendicular to the single axis.

In another embodiment, the removable holder includes a removablesource-holder for holding the fluid-source reservoir, and a removabletarget-holder for holding the fluid-target reservoir. The removablefluid source-holder and the removable target-holder are independentlymovable in any direction within separate planes separated by a distancealong the single axis.

The fluidics handling system can also include at least one removablepipette-tip holder for holding pipette tips. The pipette-tip holder caninclude an apparatus for precisely and repeatably positioning thepipette-tip holder on a pipette-tip holder support, the pipette-tipholder movable in a plane that is perpendicular to the single axis anddisplaced from the planes of motion of the removable fluid source-holderand the removable target-holder.

In another embodiment, at least one of the fluid-source reservoir, thefluid-target reservoir, and the removable holder include a trackingdevice. The tracking device includes at least one of a barcode, aradio-frequency identification (RFID) transponder programmed with aunique code readable by an RFID interrogator by non-contact means, and asemi-conductor memory device programmed with a unique code. The uniquecode is readable by at least one of an electric sensor and an externalsensor that is in communication with the semi-conductor memory devicethrough at least one of optical, infra-red, and radio-frequencycommunication. In a further embodiment, the fluidics handling systemincludes a means for locally storing and updating information on atleast one of the fluid-source reservoir, the fluid-target reservoir, andthe removable holder. The means includes at least one of a barcode; aradio-frequency identification (RFID) transponder that is dynamicallyprogrammable onto the at least one of the fluid-source reservoir, thefluid-target reservoir, and the removable holder, the transponderreadable by an RFID interrogator by non-contact means; and asemi-conductor memory device that is dynamically programmable onto theat least one of the fluid-source reservoir, the fluid-target reservoir,and the removable holder, the semi-conductor memory device readable byat least one of electrical contact and an external sensor that is incommunication with the semi-conductor memory device through at least oneof optical, infra-red, and radio-frequency communication.

In one embodiment, overall removable holder and holder-support systemposition accuracy is within ±0.02″ in x, y, and z-axes. In a preferredembodiment, overall removable holder and holder-support system positionaccuracy is within ±0.002″ in a z-axis and within ±0.01″ in x and yaxes. In a more preferred embodiment, overall removable holder andholder-support system position accuracy is within ±0.0002″ in a z-axisand within ±0.001″ in the x and y axes.

In another aspect, the invention relates to a method of transferringfluid from a source reservoir to a target reservoir. The method includesproviding a dispensing head for aspirating and dispensing fluids, thedispensing head constrained to move along a single axis. The method alsoincludes providing a fluid-source holder for holding a source-reservoir,the fluid-source holder constrained to move in a plane substantiallyperpendicular to the single axis, the fluid-source holder including anapparatus for repeatably positioning the fluid-source holder on afluid-source holder support. In addition the method includes the step ofproviding a fluid-target holder for holding a fluid-target reservoir,the fluid-target holder constrained to move in a plane substantiallyperpendicular to the single axis, a plane of motion of the fluid-targetholder displaced from a plane of motion of the fluid-source holder, thefluid-target holder including an apparatus for repeatably positioningthe fluid-target holder on a fluid-target holder support. The steps ofmoving the fluid-source holder to position the source-reservoir beneaththe dispensing head, lowering the dispensing head and aspirating fluidfrom the source-reservoir, raising the dispensing head, moving thefluid-target holder to position the fluid-target reservoir beneath thedispensing head, lowering the dispensing head and dispensing the fluidinto the fluid-target reservoir are also included in the method.

In another aspect, the invention relates to a microarrayer assembly fordepositing minute droplets of fluid on a substrate. The microarrayerincludes a deposit element for depositing minute droplets of fluid ontoa surface of a substrate and a removable substrate-holder for holding atleast one substrate, the substrate-holder including an apparatus forprecisely and repeatably positioning the substrate-holder on asubstrate-holder support.

In one embodiment, the apparatus for precisely and repeatablypositioning the substrate-holder on the substrate-holder supportincludes a first element for mating with a first datum disposed on thesubstrate-holder support, and for restricting movement of the removablesubstrate-holder along an x-axis and a mutually orthogonal y-axis anddefining a first point in a z-axis, the z-axis mutually orthogonal tothe x-axis and the y-axis. The apparatus also includes a second elementfor mating with a second datum disposed on the substrate-holder support,and for at least partially locating the removable substrate-holder alongat least one of the x-axis and the y-axis while defining a second pointin the z-axis. A third element is also included in the apparatus formating with a third datum disposed on the substrate-holder support anddefining a third point in the z-axis. In one embodiment, the firstelement forms a conical recess for mating with the first datum. Inanother embodiment, at least two of the elements are adjustable alongthe z-axis.

In one embodiment, the substrate-holder support further includes a datumplane defined by at least three datums including a first datum forengaging the substrate-holder and for restricting movement of thesubstrate-holder along an x-axis and a mutually orthogonal y-axis, anddefining a first point in a z-axis, the z-axis mutually orthogonal tothe x-axis and the y-axis. A second datum is also included for engagingthe substrate-holder and for at least partially locating thesubstrate-holder along at least one of the x-axis and the y-axis anddefining a second point in the z-axis. The third datum is provided forengaging the substrate-holder and for defining a third point in thez-axis. In one embodiment, the first datum includes at least a portionof a sphere for engaging a conical recess formed in thesubstrate-holder. In another embodiment the second datum is engageablewith a linear recess formed in the substrate-holder.

The microarrayer assembly in other embodiments includes a removablefluid-reservoir for holding at least one fluid, the removablefluid-reservoir including an apparatus for repeatably positioning theremovable fluid-reservoir on a fluid-reservoir holder support. In oneembodiment, the microarrayer assembly also includes a removablefluid-reservoir holder for holding the removable fluid-reservoir, thefluid-reservoir holder including an apparatus for precisely andrepeatably positioning the removable fluid-reservoir holder on thefluid-reservoir holder support. In one embodiment, the fluid-reservoirholder support moves in unison with the substrate-holder support.

In one embodiment, the apparatus for repeatably positioning theremovable fluid-reservoir holder on the fluid-reservoir holder supportincludes a first element for mating with a first datum disposed on thefluid-reservoir holder support, and for restricting movement of theremovable fluid-reservoir holder along an x-axis and a mutuallyorthogonal y-axis and defining a first point in a z-axis, the z-axismutually orthogonal to the x-axis and the y-axis. The apparatus alsoincludes a second element for mating with a second datum disposed on thefluid-reservoir holder support, and for at least partially locating theremovable fluid-reservoir holder along at least one of the x-axis andthe y-axis while defining a second point in the z-axis. A third elementis also included in the apparatus for mating with a third datum disposedon the fluid-reservoir holder support and defining a third point in thez-axis. In one embodiment, the first element forms a conical recess formating with the first datum. In another embodiment, at least two ofelements are adjustable along the z-axis.

In yet another embodiment in accordance with the invention, thefluid-reservoir holder support includes a first datum for engaging theremovable fluid-reservoir and for restricting movement of the removablefluid-reservoir along an x-axis and a mutually orthogonal y-axis, anddefining a first point in a z-axis, the z-axis mutually orthogonal tothe x-axis and the y-axis. A second datum is also included for engagingthe removable fluid-reservoir and for at least partially locating theremovable fluid-reservoir along at least one of the x-axis and they-axis and defining a second point in the z-axis. The fluid-reservoirholder also includes a third datum for engaging the removablefluid-reservoir and defining a third point in the z-axis. In oneembodiment, the first datum includes at least a portion of a sphere forengaging a conical recess disposed on the removable fluid-reservoir. Inyet another embodiment, the second datum is engageable with a linearrecess disposed on the removable fluid-reservoir.

The microarrayer assembly can also include a variety of other features.For instance, in one embodiment, the deposit element comprises a solidpin. The microarrayer assembly can include a fluid-reservoir storage andan apparatus for moving a fluid-reservoir between the fluid-reservoirstorage and the fluid-reservoir holder support. In addition, a sensorcan be included to determine presence of a fluid-reservoir in a bay ofthe fluid-reservoir storage. Similarly, the microarrayer assembly caninclude a substrate-holder storage and an apparatus for moving asubstrate-holder between the substrate-holder storage and thesubstrate-holder support. A sensor can also be included to determinepresence of a substrate-holder in a bay of the substrate-holder storage.In one embodiment, the substrate-holders are moved from thesubstrate-holder storage to the substrate-holder support by moving thesubstrate-holder in a vertical direction within the substrate-holderstorage to dispose the substrate-holder on the substrate holder supportand moving the substrate-holder support in a horizontal direction toretract the substrate-holder support from the substrate-holder storage.In a further embodiment, the removable substrate-holders are at leastone of removed from and added to the substrate-holder storage duringactive fluid capture and droplet deposition operations.

In one embodiment, the deposit element is constrained to move along az-axis and the substrate-holder is constrained to move in a planesubstantially perpendicular to the z-axis when disposed beneath thedeposit element. In yet another embodiment, the deposit element isconstrained to move along a z-axis and the fluid-reservoir isconstrained to move in a plane substantially perpendicular to the z-axiswhen disposed beneath the deposit element. In a further adaptation, thefluid-reservoir plane of motion is parallel to and displaced from thesubstrate-holder plane of motion when disposed beneath the depositelement. In a further embodiment, the fluid-reservoir is moveableindependently of but in coordination with the substrate-holder and thedeposit element.

In one embodiment, the removable fluid-reservoir is a multi-well platehaving 96 wells or a multiple thereof. The substrate, in anotherembodiment, may also include a multi-well plate.

In a further adaptation, the microarrayer assembly includes a firstprinthead and a second printhead, each printhead for holding at leastone deposit element, where the first printhead and the second printheadare optionally arranged for moving independently of each other inseparate parallel axes.

In another embodiment, the removable substrate-holder includes a topsurface and a bottom surface spaced from the top surface, an apertureextending at least partially between the top surface and the bottomsurface, an intersection of the aperture and the top surface defining aperimeter and at least three reference points proximate the perimeterand defining a reference plane, the reference points for engaging a topsurface of the substrate when the substrate is disposed in the aperture.Means may be included in the removable-substrate holder to bias thesubstrate against the at least three reference points. In yet anotherembodiment, the removable substrate-holder includes a removablesubstrate-mounting fixture, the substrate-mounting fixture including alocking element for securing the substrate-mounting fixture into theremovable substrate-holder, the substrate-mounting fixture for holdingat least one substrate.

In one embodiment of the microarrayer assembly, a sensor is included tomeasure a distance from the deposit element to a top surface of thesubstrate. In addition, a motion control system may be included todynamically adjust a motion of the deposit element in response to thesensor measurement to deposit the minute droplet of fluid onto thesubstrate without the deposit element contacting the substrate.

In a further embodiment, the microarrayer assembly includes a barcodereader for optically sensing labels secured to at least one of thesubstrate-holder, the substrate, the fluid-reservoir holder, and thefluid-reservoir. In another embodiment at least one of thesubstrate-holder and the fluid-reservoir holder further comprise atracking device. The tracking device includes at least one of a barcode,a radio-frequency identification (RFID) transponder programmed with aunique code readable by an RFID interrogator by non-contact means, and asemi-conductor memory device programmed with a unique code. The uniquecode disposed on the semi-conductor memory device is readable by atleast one of an electric sensor and an external sensor that is incommunication with the semi-conductor memory device through at least oneof optical, infra-red, and radio-frequency communication. In addition,the microarrayer assembly can include a means for locally storing andupdating information on at least one of the substrate, thesubstrate-holder, the fluid reservoir, and the fluid-reservoir holder.The means includes at least one of a radio-frequency identification(RFID) transponder and a semi-conductor memory device dynamicallyprogrammable onto the at least one of the substrate, thesubstrate-holder, the fluid reservoir, and the fluid-reservoir holder.The transponder is readable by an RFID interrogator by non-contact meansand the semi-conductor memory device is readable by at least one ofelectrical contact and an external sensor that is in communication withthe semi-conductor memory device through at least one of optical,infra-red, and radio-frequency communication.

In one embodiment, overall substrate-holder and substrate-holder supportsystem position accuracy is within ±0.002″ in a z-axis and within ±0.01″in x and y axes. In a preferred embodiment, overall substrate-holder andsubstrate-holder support system position accuracy is within ±0.001″ in az-axis and within ±0.005″ in x and y axes. In a more preferredembodiment, overall substrate-holder and substrate-holder support systemposition accuracy is within ±0.0002″ in a z-axis and within ±0.001″ in xand y axes.

In another aspect, the invention relates to a microarrayer assembly fordepositing minute droplets of fluid on a substrate. The microarrayerassembly includes a plurality of deposition engines operatingcooperatively. Each deposition engine includes a deposit element fordepositing minute droplets of fluid onto a surface of a substrate and asupport for holding at least one of a substrate-holder and a fluidreservoir, the support including an apparatus for precisely andrepeatably positioning at least one of the substrate-holder and thefluid reservoir on the support.

In one embodiment, the microarrayer assembly further includes means totransfer at least one of the substrate-holder and the fluid-reservoirbetween the deposition engines. In addition, the microarrayer assemblycan include at least one hotel for storing at least one of thesubstrate-holder and a fluid-reservoir and a means to transfer at leastone of the substrate-holder and the fluid-reservoir between the hoteland at least one deposition engine.

In another aspect, the invention relates to a method for depositingminute droplets of fluid on a substrate and a microarray produced inaccordance with the method. The method includes the step of loading asubstrate-holder onto a substrate-holder support, the substrate-holderfor holding at least one substrate and the substrate-holder including anapparatus for precisely and repeatably positioning the substrate-holderon the substrate-holder support. In addition, the method includes thesteps of providing fluid to a deposit element, the deposit elementmoveable relative to the substrate-holder and transferring a droplet offluid from the deposit element to the substrate.

In one embodiment, the method also includes the step of transferring thesubstrate-holder between a substrate-holder storage and thesubstrate-holder support. The method may also include the step ofloading a fluid-source holder onto a fluid-source holder support, thefluid-source holder for holding at least one fluid source and includingan apparatus for precisely and repeatably positioning the fluid-sourceholder on the fluid-source holder support. In a further embodiment, themethod also includes the steps of transferring the fluid-source holderfrom a fluid-source holder storage to the fluid-source holder support,capturing fluid from the fluid-source with the deposit element, andtransferring the fluid-source holder from the fluid-source holdersupport to the fluid-source holder storage. In one embodiment, thedeposit element includes a solid pin.

In one embodiment, the substrate-holder support includes a first datumfor engaging the substrate-holder and for restricting movement of thesubstrate-holder along an x-axis and a mutually orthogonal y-axis, anddefining a first point in a z-axis, the z-axis mutually orthogonal tothe x-axis and the y-axis. The substrate-holder support also includes asecond datum for engaging the substrate-holder and for at leastpartially locating the substrate-holder along at least one of the x-axisand the y-axis and defining a second point in the z-axis. Further, thesubstrate-holder support includes a third datum for engaging thesubstrate-holder and defining a third point in the z-axis. In oneembodiment, the first datum includes at least a portion of a sphere forengaging a conical recess disposed on the substrate-holder. In anotherembodiment, the second datum is engageable with a linear recess disposedon the substrate-holder.

In one embodiment, the substrate-holder includes a first element formating with a first datum, and for restricting movement of thesubstrate-holder along an x-axis and a mutually orthogonal y-axis anddefining a first point in a z-axis, the z-axis mutually orthogonal tothe x-axis and the y-axis. The substrate-holder further includes asecond element for mating with a second datum, and for at leastpartially locating the substrate-holder along at least one of the x-axisand the y-axis while defining a second point in the z-axis. In addition,the substrate-holder includes a third element for mating with a thirddatum and defining a third point in the z-axis. In one embodiment, thefirst element forms a conical recess for mating with the first datum.

In yet another embodiment, the method may include the step of moving afluid reservoir to a position beneath the deposit element, the depositelement constrained to travel substantially along a vertical axis andthe fluid reservoir being constrained to travel in a plane substantiallyperpendicular to the vertical axis when disposed beneath the depositelement. The steps of lowering the deposit element to capture fluid fromthe fluid reservoir, raising the deposit element relative to the fluidreservoir, moving the substrate-holder to a position beneath the depositelement, the substrate-holder constrained to travel in a plane parallelsubstantially perpendicular to the vertical axis when disposed beneaththe deposit element and lowering the deposit element to deposit thefluid on the substrate can also be included in the method.

In another aspect, the invention relates to a method of depositingdroplets of fluid on a substrate and a microarray produced in accordancewith the method. The method includes the step of moving a fluidreservoir to a position beneath a printhead, the printhead beingconstrained to travel along a vertical axis and the fluid reservoirbeing constrained to travel within a plane substantially perpendicularto the vertical axis when disposed beneath the printhead. Also includedin the method are the steps of lowering the printhead to capture fluidfrom the fluid reservoir, raising the printhead relative to the fluidreservoir and moving a substrate to a position beneath the printhead,the substrate constrained to travel within a plane parallel to, butdisplaced from, the plane of motion of the fluid reservoir when disposedbeneath the printhead. The method also includes the step of lowering theprinthead to deposit the fluid on the substrate.

In one embodiment, the method further includes the step of moving thefluid reservoir away from the axis of motion of the printhead prior todepositing the fluid on the substrate. In a further embodiment, the stepof moving the fluid reservoir to a position beneath the printheadincludes moving the substrate in tandem with the fluid reservoir. In yetanother embodiment, the step of moving the substrate beneath theprinthead precedes the step of moving the fluid reservoir away from theaxis of motion of the printhead.

In another aspect, the invention relates to a method of depositingminute droplets of fluid on a substrate. The method includes the step ofarranging a plurality of deposition engines cooperatively, eachdeposition engine including a deposit element for depositing minutedroplets of fluid onto a surface of a substrate. The method alsoincludes the step of transferring at least one holder between thedeposition engines, the holder for holding at least one of a substrateand a fluid-reservoir, the holder including an apparatus for preciselyand repeatably positioning the holder on a support.

In one embodiment, each deposition engine comprises a plurality ofprintheads. In another embodiment, the method includes the step oftransferring at least one holder from a hotel to at least one depositionengine. The holders may be removed from and added to the hotel duringactive fluid capture and droplet deposition operations.

In another aspect, the invention relates to a microarrayer assembly fordepositing minute droplets of fluid on a substrate. The microarrayerincludes a printhead for depositing fluids on the substrate, a fluidreservoir including at least one well for supplying fluid to theprinthead, and a sensor for measuring depth of fluid in the at least onewell.

In another aspect, the invention relates to a method for depositingminute droplets of fluid on a substrate. The method includes the step ofmoving a fluid reservoir to a position beneath a first printhead, thefirst printhead including at least one deposition element andconstrained to move along a vertical axis, the fluid reservoirconstrained to move in a plane substantially perpendicular to thevertical axis when disposed beneath the first printhead. Also includedin the method are the steps of moving the printhead relative to thefluid reservoir to dip the deposition element into the fluid reservoir,raising the first printhead relative to the fluid reservoir, and movinga substrate beneath the first printhead while simultaneously moving thefluid reservoir beneath a second printhead, the substrate constrained tomove in a plane parallel to, but displaced from, the plane of motion ofthe fluid reservoir. The steps of lowering the first printhead todeposit a fluid droplet on the substrate and lowering the secondprinthead to capture fluid from the fluid reservoir, and raising thefirst printhead and the second printhead above the planes of motion ofthe substrate and the fluid reservoir are also included in the method.The method also includes the steps of moving the substrate under thesecond printhead while simultaneously moving the fluid reservoir to aposition beneath the first printhead, and lowering the second printheadto deposit a fluid droplet on the substrate and lowering the firstprinthead to capture fluid from the reservoir.

In one embodiment, the method includes the step of moving the substrateunder at least one of the first printhead and second printhead prior tomoving the fluid reservoir away from at least one of the first printheadand the second printhead.

In another aspect, the invention relates to a method for depositingbiological fluid samples onto a substrate to reduce non-specific bindingin undesired locations on the substrate. The method includes the step ofproviding a substrate including a surface resistant to non-specificbinding of biological material. A second step included in the method isdepositing a first fluid droplet onto the substrate, the first fluiddroplet including a binding agent that is bindable with the substrateand that is bindable to a biological material through at least one ofelectrostatic, covalent and chemical binding. The method also includesthe step of depositing a second fluid droplet onto the depositionlocation of the first fluid droplet, the second fluid droplet includinga biological material for binding with the binding agent.

In one embodiment, the method includes the step of depositing additionalfluid droplets on the deposition location of the first fluid droplet todeactivate the binding properties of any unbound binding agent andbiological material remaining from the first droplet and the seconddroplet.

These and other objects, along with advantages and features of thepresent invention herein disclosed, will become apparent throughreference to the following description, the accompanying drawings, andthe claims. Furthermore, it is to be understood that the features of thevarious embodiments described herein are not mutually exclusive and canexist in various combinations and permutations.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference characters generally refer to the sameparts throughout the different views. In addition, the drawings are notnecessarily to scale, emphasis instead generally being placed uponillustrating the principles of the invention. In the followingdescription, various embodiments of the present invention are describedwith reference to the following drawings, in which:

FIG. 1 is a schematic perspective view of a microarrayer in accordancewith one embodiment of the invention;

FIG. 2A is a schematic perspective view of a printhead including a pinsupport assembly in accordance with one embodiment of the invention;

FIG. 2B is a schematic cross-sectional view of the pin support assemblyof FIG. 2A taken at line 2B-2B in FIG. 2A;

FIG. 3 is a schematic perspective view of a top referencingsubstrate-holder in accordance with one embodiment of the invention;

FIGS. 4A-4B are schematic perspective views of a top-referencedsubstrate-holder and a detachable substrate-mounting fixture forsecuring the substrate to the substrate-holder in accordance with oneembodiment of the invention.

FIG. 4C is a schematic exploded perspective view of the top-referencedsubstrate-holder and the detachable substrate-mounting fixture of FIGS.4A-4B.

FIG. 5A is a schematic bottom view of a substrate-holder includinginserts for mating with datums disposed on a substrate-holder support inaccordance with one embodiment of the invention;

FIG. 5B is a schematic bottom view of a substrate-holder includinginserts for mating with datums disposed on a substrate-holder support inaccordance with one embodiment of the invention;

FIG. 5C is a schematic bottom view of a substrate-holder includinginserts for mating with datums disposed on a substrate-holder support inaccordance with one embodiment of the invention;

FIG. 5D is a schematic exploded perspective view of a substrate-holderincluding parallel grooves for mating with precision adjustment screwsdisposed on a substrate-holder support in accordance with one embodimentof the invention;

FIG. 5E is a schematic perspective view of a substrate-holder supportstructure in accordance with one embodiment of the invention;

FIG. 6A is a schematic exploded perspective view of a substrate-holdersupport including datums for engaging reference surfaces disposed on asubstrate-holder in accordance with one embodiment of the invention;

FIG. 6B is a schematic exploded perspective view of a substrate-holdersupport including datums for engaging reference surfaces disposed on asubstrate-holder in accordance with one embodiment of the invention;

FIG. 6C is a schematic exploded perspective view of a substrate-holdersupport including inserts for engaging datums disposed on asubstrate-holder in accordance with one embodiment of the invention;

FIG. 7 is a schematic exploded perspective view of a fluid-reservoirholder including inserts for engaging datums disposed on afluid-reservoir holder support in accordance with one embodiment of theinvention;

FIG. 8 is a schematic exploded perspective view of a fluid-reservoirholder including reference surfaces for engaging datums disposed on afluid-reservoir holder support in accordance with one embodiment of theinvention;

FIG. 9 is a schematic perspective view of a microarrayer architecture inaccordance with one embodiment of the invention;

FIG. 10 is a schematic perspective view of a microarrayer architecturein accordance with one embodiment of the invention;

FIG. 11 is a schematic perspective view of a microarrayer architecturein accordance with one embodiment of the invention;

FIG. 12 is a schematic perspective view of a microarrayer architecturein accordance with one embodiment of the invention;

FIG. 13 is a schematic block diagram illustration of a multi-enginemicroarrayer assembly for use with non-aspirating ink-jet dispensers inaccordance with one embodiment of the invention;

FIG. 14 is a schematic block diagram illustration of a multi-enginemicroarrayer assembly for use with aspirating deposit elements inaccordance with one embodiment of the invention;

FIG. 15 is a schematic perspective view of a tissue arrayer inaccordance with one embodiment of the invention; and

FIG. 16 is a schematic perspective view of a fluidics robot inaccordance with one embodiment of the invention.

DETAILED DESCRIPTION

Embodiments of the present invention are described below. It is,however, expressly noted that the present invention is not limited tothese embodiments, but rather the intention is that variations,modifications, and equivalents that are apparent to the person skilledin the art are also included. The detailed description is written inthree parts. The first part discusses Microarrayers, the second partdiscusses Tissue Arrayers, and the third part discusses Fluidics Robots.Since the term “microarray” is often used in the art to describe both anarray of fluid samples and an array of tissue samples, a distinction interminology is used in this disclosure. The terms “microarray” or“spotted microarray” are used to refer to an array of samples depositedin a fluid state upon a substrate in the form of minute fluid droplets.The terms “tissue microarray” or “tissue array” are used to refer to anarray of tissue samples deposited in semi-solid or solid form.

Similarly, the term “microarrayer”, in this disclosure will be used torefer to a device for producing microarrays of fluid droplets. The terms“arrayer” and “spotter” may be used synonymously for the termmicroarrayer. The term “tissue arrayer”, in this disclosure, will beused to refer to a device for producing tissue arrays.

1) Microarrayers

With reference to FIG. 1, in one embodiment of a microarrayer assembly10 in accordance with the invention, the microarrayer assembly 10includes a printhead 12 which holds a plurality of deposit elements 14.Also included in the microarrayer assembly 10 are substrates 16 whichare held on a substrate-holder 18 which is in turn mounted on a platen89. A fluid reservoir 20, for instance a microplate or microtiter plate,is also included in the microarrayer assembly 10. The fluid reservoir 20is held on a fluid reservoir holder 21 and, in turn, the fluid reservoirholder 21 is held on the platen 89. In one embodiment, the printhead 12and the platen 89 are each mounted on motion stages (not shown). Themotion stages enable the printhead 12 and the platen 89 to moverelatively in relation to each other so that the deposit elements 14 canacquire fluid from the fluid reservoir 20 and deposit the fluid on thesubstrates 16.

a) Deposit Elements

A variety of deposit elements 14 may be used in accordance with theinvention including ink-jet dispensers, pens, quill pins, and solidpins. Ink jet dispensers 14 eject drops onto a substrate 16 using, forinstance, a piezoelectric crystal which deforms in response to a voltageto squeeze a minute droplet of fluid from a minute orifice in thedispenser. Fluid samples to be dispensed from an ink jet device 14 areeither fed directly to the device, for example via tubing, or,alternatively, the sample fluid can be aspirated into the ink jet device14 from a fluid reservoir 20.

In embodiments using pen printing, a pen-like device 14 such as a narrowcapillary tube is first dipped into the fluid reservoir 20 to aspiratefluid, and then used to deposit a fluid droplet upon the substrate 16 byapplying pressure to the fluid within the capillary.

Quill-pin printing embodiments in accordance with the invention use asplit pin 14 or pin with a slit near its tip. The quill pin 14 is firstdipped in the fluid reservoir 20 to capture fluid in the slit betweenthe two segments of the pin 14. This local fluid reservoir in the slitis then used to re-supply the tip of the pin when the pin 14 is touchedor tapped upon the surface of the substrate 16.

Solid pins 14 may also be used as the deposit element 14 in accordancewith other embodiments of the invention. When used as the depositelement 14, solid pins with tip diameters between about 25 micrometersto about 700 micrometers may be used, and in another embodiment, solidpins having diameters between about 70 micrometers to about 300micrometers may be used. The tip of the pin is dipped into a fluidreservoir 20 (for instance into fluid held within a well of a microplate20) from which the pin 14 is then withdrawn such that a droplet of fluidis captured on the tip of the pin 14. The pin 14 is then moved,relatively, to touch the tip of the pin 14, or to touch the fluiddroplet adhered to the tip of the pin 14, onto a substrate 16 andthereby transfer some of the fluid to the substrate 16.

With reference to FIGS. 2A-2B, solid pins 14 are shown held within apin-support assembly 24 that maintains precise positioning of a tip 26of the pin 14 in a horizontal plane while allowing compliance in avertical dimension. The solid pins 14 in various embodiments, arestepped pins and tapered pins. The pin support assembly 24 includesupper holes 28 and lower holes 30 through which the pins 14 extend. Theupper holes 28 are formed slightly larger than the width (the diameterof the pin, if of circular cross-section) of the pin's upper sectionsuch that the pin 14 can move vertically within the pin support assembly24, but is constrained to move minimally in the horizontal plane. Asillustrated, the lower holes 30 each include a conical seat 32 thatmatches a similar profile formed on the pin 14. The conical seat 32serves both to a) provide a lower restraint to vertical motion of thepin 14, thereby defining the vertical location of the tip 26 of the pin14 of known length and proportions, and b) to precisely locate the lowersection of the pin 14, and thereby the pin tip 26, in the horizontalplane. In one embodiment, the pin 14 is pushed against the conical seat32 by the weight of the pin 14 alone. In another embodiment, a biasingmeans, such as a vertically acting spring is used to thrust the conicalsection of the pin 14 onto the seat 32. It will be appreciated thatother techniques for maintaining the position of the pin tip 26 may beused, and the above example should not be considered limiting.

With reference to FIGS. 1-2B, a multiplicity of deposit elements 14 areheld in the printhead 12 which greatly enhances the rate at whichdroplets may be deposited on a substrate 16. Typically, the depositelements 14 are spaced apart at a distance corresponding to thecenter-to-center well spacing of the multi-well fluid reservoir 20 beingused, such as a microplate 20 having 24, 48, 96, 384, 1536 or 3456wells, or a microplate having a number of wells being a multiple of anyof these numbers. One advantage of the use of solid pins 14 compared toquill-pins or pens is that their relatively narrow tips 26 allows theirpenetration into small, high density wells, such as those in standard1536 and 3456 microplates 20. Similarly, the narrow bodies of solid pinsenable printheads 12 to contain a higher density of pins 14 within agiven area. Solid-pin printheads 12 of 192 pins or more can readily beused in combination with 1536-well microplates 20.

Once the deposit elements 14 are mounted in the printhead 12, planaradjustments of the tips 26 are desirable to bring the plane of the tips26 parallel to a plane of the substrates 16. For example, precisionadjustments to the pitch, roll and yaw between fixed reference elementsof the printhead 14 and the pin-support assembly 24 may be made withadjustment screws 33.

b) Substrates and Substrate Holders

Microarrays of fluid droplets can be spotted on a wide variety ofsubstrates 16. In one embodiment, the substrate 16 is in the form of aglass slide, such as a microscope slide. The substrate 16, in anotherembodiment, is a multi-well plate such as a micro-titer plate withflat-bottomed wells. The benefit of using such a multi-well plate isthat the fluids spotted on the flat bottom of each well can beindependently assayed. This is of significant value for applicationssuch as drug discovery, high-throughput screening and toxicogenomics.The well-plate format for the substrate 16 is well suited toapplications requiring a multiplicity of parallel tests on a limitednumber of fluid samples, typically up to several thousand in number.

In other embodiments in accordance with the invention, the substratesmay be selected from a variety of materials and forms, all of which areincluded within the scope of the present invention. Such materialsinclude, but are not limited to, metal, plastic, nylon, semiconductorand ceramic materials, glass plates, clear or glass-bottomed well platesor similar multi-well structures allowing for further independentchemical or biological processing.

In another embodiment, a top surface 17 of the substrate 16 is coatedwith a material that will bind biological molecules. In one embodiment,the coating has hydrophobic properties to minimize the spreading of thedroplet over the top surface 17 of the substrate 16. Many coatings havebeen developed for microarray substrates 16 and will be familiar tothose skilled in the art.

As mentioned earlier, a limitation of the prior art is that substratemounting arrangements have been bottom-referenced on a platen, i.e. thesubstrate such as a glass slide, is mounted such that its bottom surfacerests upon the top surface of the platen. Variability in the thicknessof the substrates can create uncertainty in the height of the surfaceupon which fluid droplets will be deposited and can cause undesirablevariations in droplet deposition from substrate to substrate.

With reference to FIG. 1, in one embodiment in accordance with theinvention, this limitation is overcome by providing a distancemeasurement sensor 34 (in one embodiment the distance measurement sensoris mounted to the printhead 12 to minimize uncertainties in absoluteposition) to accurately measure the distance between the printhead 12and the top surface 17 of the substrate 16 onto which fluid droplets areto be deposited. Precision motion control elements can then be employedto adjust the relative distance between the tips 26 of the depositelements 14 and the substrate 16 to effect contact of the droplet andthe top surface 17 of the substrate 16. The distance measurement sensor,for example, may be:

-   -   a) capacitive, wherein the change in capacitance as an element        approaches an object or surface is sensed to measure distance,    -   b) inductive, wherein the change in inductance as an element        approaches an object or surface is sensed to measure distance,    -   c) conductive, wherein conduction of an electric current or        signal (continuous or alternating current) is either established        or broken when an element touches an object or surface,    -   d) magnetic, wherein the change in magnetic flux as an element        approaches an object or surface is sensed to measure distance,    -   e) optical, including, but not limited to i) laser        interferometry distance measurement, ii) optical switching (in        which an optical beam is either established or broken as a        result of physical contact of an element or an optical beam with        an object or surface), iii) optical displacement sensing, in        which the distance to a surface is measured by measuring the        displacement of a beam that is reflected from that surface at an        angle other than normal incidence, or    -   f) radar, sonar or laser-radar based, with distance measurement        using pulsed transmissions or modulated continuous-wave        transmissions.

In another embodiment, variability in the thickness of the substrates 16is overcome by top referencing the substrates 16 in the substrate-holder18. With reference to FIG. 3, an arrangement for top-referencing asubstrate 16 in a substrate-holder 18 is illustrated. The top surface 36of the substrate-holder 18 (or at least those parts of the top surface36 around the locations where the substrates 16 are mounted) is machinedor constructed to be precisely co-planar. Precisely machined brackets 38are coupled to the machined top surface of the substrate-holder 18 suchthat portions 40 of the brackets 38 protrude over a recess in which thesubstrates 16 are disposed and present a three-point support to define aplane against which the top surface 17 of the substrate 16 rests. Inthis way, the top surface 17 of each substrate 16 will be substantiallycoplanar with the top surface 17 of every other substrate 16 on thesubstrate-holder 18, regardless of the individual thicknesses of thesubstrates 16. Resilient spring clips press the substrate 16 against theprotruding portions 40 of the bracket 38 from below.

Alternative methods for top-referencing the substrate 16 in thesubstrate-holder 18 exist. For example, rather than using brackets, thereference surface 36 onto which the top surface 17 of the substrate 16is pressed could be continuous and protrude over the aperture whichreceives the substrate 16. All such alternative realizations that havethe effect of precisely locating the top surface 17 of the substrate 16in the same plane are included within the scope of the presentinvention.

With reference to FIGS. 4A-4C, in another embodiment, the substrate 16is initially mounted on a detachable substrate-mounting fixture 42 thatis, in turn, coupled to the substrate-holder 18 such that the topsurface 17 of the substrate 16 is pressed upon the aforementionedreference surfaces 40 of the substrate-holder brackets 38. Thesubstrate-holder 18 includes recesses 27 designed to accept theslide-mounting fixture 42 with the substrate 16 mounted upon it. Thesubstrate 16 is referenced in the substrate-mounting fixture 42 at oneend and on one side by fixed tabs 44 that do not extend above the topsurface of the substrate 16. The substrate 16 is held against the tabs44 by a spring clip 46 which exerts pressure on the side of thesubstrate 16, but does not extend above the top surface 17 of thesubstrate 16. With reference to FIG. 4C, a retaining device 48, withprojections 49 for twisting the device 48 by finger action, is mountedon the bottom surface of the substrate-mounting fixture 42. Theretaining device 48 is free to rotate around a vertical z-axis. Theretaining device 48 is mounted on a compression spring (not shown) whichpushes it away from the bottom surface of the substrate-mounting fixture42 against a restraint which limits its motion away from thesubstrate-mounting fixture 42. Two bridges 50 with cam surfaces 51formed on upper surfaces facing the substrate-holder 18 are firmlycoupled to the substrate-holder 18 at either side of the recess 27. Thesubstrate-mounting fixture 42 is inserted into the recess 27 in thelower side of the substrate-holder 18 until the top surface 17 of thesubstrate 16 contacts the reference surfaces 40 of the bracket 38 (thereference surfaces 40 being coplanar for all slide-mounting fixturerecesses). The retaining device 48 is rotated, such that its projections49 engage and slide over the cam surfaces 51 of the bridges 50. Thisaction compresses the spring and causes the top surface 17 of thesubstrate 16 to press firmly against the reference surfaces 40 of thesubstrate-holder bracket 38. Detents at the centers of the cam surfaces51 provide a position in which the retaining device 48 securely rests ina “locked” position.

The repeatable, accurate, top-referenced mounting structures of thepresent invention enable non-contact deposition without a substrateheight position measurement sensor 34. Alternatively if such a sensor 34is beneficial, a single measurement may suffice for all substrates 16 onthe substrate-holder 18.

With reference to FIG. 5A, in another embodiment, the substrate-holder18 includes an apparatus for mounting the substrate-holder 18 on asubstrate-holder support 19, such that the substrates 16 may beprecisely positioned repeatedly in a defined plane and at a definedlocation in that plane.

Reference surfaces are machined or formed on three inserts 54 a, 54 b,and 54 c that are securely affixed in two corners of thesubstrate-holder 18 and at a mid-point on the far side of thesubstrate-holder 18 as illustrated. In one embodiment, the insert 54 aincludes a conically-shaped reference surface 55. The insert 54 a issecured into the substrate-holder 18, for instance, by screwing theinsert 54 a into the substrate-holder 18, and then optionally fasteningthe insert 54 a into position using an adhesive. The insert 54 a, in oneembodiment, is made from a hardened metal. In other embodiments,materials that are machinable, while also being non-deformable may beused. The insert 54 a is engageable with a datum disposed on asubstrate-holder support 19 to restrict movement of the substrate-holder18 along an x-axis and a mutually orthogonal y-axis and defining a firstpoint in a z-axis, where the z-axis is mutually orthogonal to the x-axisand the y-axis.

The insert 54 b in the adjacent corner of the substrate-holder includesa V-groove reference surface 56. The insert 54 b is inserted into thesubstrate-holder 18 in one embodiment such that the axis of the V-groovepasses through the apex of the conical surface 55. The insert 54 b maybe secured to the substrate-holder 18 via a dowel pin 58 and adhesive.The insert 54 b can be made from a hardened metal. The insert 54 b isengageable with a second datum 64 disposed on the substrate-holdersupport 19 to locate the substrate-holder 18 along at least one of thex-axis and the y-axis while defining a second point in the z-axis.

The insert 54 c on the far end of the substrate-holder 18 in oneembodiment includes a flat (horizontal) surface 60. The insert 54 c maybe secured to the substrate-holder 18 via a screw thread and adhesive.The insert 54 c may be made, for example, from a hardened metal. Theinsert 54 c is engageable with a third datum disposed on thesubstrate-holder support 19 and defines a third point in the z-axis.

With reference to FIG. 6A, as mentioned, the reference surfaces 55, 56,60 of the inserts 54 a, 54 b, 54 c are designed to rest in contact withdatums 64 disposed on the substrate-holder support 19. The datums aredisposed on the substrate-holder support such that they align with theinserts 54 a, 54 b, and 54 c disposed on the substrate-holder 18. In theillustrated embodiment, the datums 64 include hemispherical surfaces. Inone embodiment, the hemispherical surfaces are provided by the top sidesof hardened ball bearings mounted in precision seats set inoptical-plane adjustment screws. The adjustment screws can be adjustedin height to set the substrate-holder 18 in the desired plane, and thenlocked in place.

In another embodiment, at least one datum 64 includes at least a portionof a spherical surface. In a further embodiment, at least one datum 64includes a point formed by a pin. In another embodiment, the datums 64are disposed on the substrate-holder 18 and the inserts are disposed onthe substrate-holder support 19 (FIG. 6C).

In use, when the reference surfaces 55, 56, 60 of the inserts 54 a, 54b, 54 c are received on the datums 64, the datums 64 and the referencesurfaces 55, 56, 60 locate the substrate-holder 18 in a selected plane.Further, if the substrate-holder 18 is removed from the substrate-holdersupport 19, and then re-seated on the substrate-holder support 19, thesubstrate-holder 18 will locate itself in the identical plane and in theidentical location in the plane. In addition, any substrate-holder 18with inserts 54 a, 54 b, 54 c with reference surfaces set in the samepositions and at the same heights (the heights are adjustable on two ofthe inserts 54 a and 54 c) will be accurately located in the sameposition in the same plane with respect to the substrate-holder support19. This embodiment therefore permits a series of substrate-holders 18to be sequentially loaded into the microarrayer assembly 10 without theloss of positional accuracy in locating the substrate-holders 18 on thesubstrate-holder support 19. In the illustrated embodiment, gravityloading is sufficient to firmly and accurately seat the substrate-holder18 on the datums 64 of the substrate-holder support 19. In anotherembodiment, additional means for restraining the substrate-holder 18against the datums 64 of the substrate-holder support 19 may beutilized. For instance, magnetic, electromagnetic, electrostatic, vacuumor mechanical means could be used for this purpose. In combination, thereference surfaces 55, 56, and 60 disposed on the substrate-holder 18,the datums 64 on the substrate-holder support 19, along with the topreferencing of substrates 16 in a substrate-holder 18 provide anapparatus for accurately locating the top surface 17 of the substrates16 in a known position in a known plane in the microarrayer assembly 10.

The capability to load substrate-holders 18 into and out of amicroarrayer assembly 10 while maintaining positional accuracy of thesubstrates 16 in three dimensions serves to separate the choice offunctional design of the microarrayer deposition apparatus from theselection of the substrate-handling capacity of the microarrayerassembly 10.

In one embodiment, overall substrate-holder and substrate-holder supportsystem position accuracy is within ±0.002″ in the z-axis and within±0.01″ in the x and y axes. In a preferred embodiment, overallsubstrate-holder and substrate-holder support system position accuracyis within ±0.001″ in the z-axis and within ±0.005″ in the x and y axes.In a more preferred embodiment, overall substrate-holder andsubstrate-holder support system position accuracy is within ±0.0002″ inthe z-axis and within ±0.001″ in the x and y axes.

With reference to FIGS. 5B and 6B, in another embodiment, referencesurfaces are machined or formed on four hardened metal inserts 54 a, 54b, 54 c and 54 d that are securely affixed in the corners of thesubstrate-holder 18. In one embodiment, the insert 54 a includes aconically—shaped reference surface 55. Additional hardened metal inserts54 b and 54 d in the two adjacent corners of the substrate-holderinclude V-groove reference surfaces 56, 57. The two V-groove referencesurfaces 56, 57 are oriented with the axes of their grooves passingthrough the apex of the conical surface 55. The inserts 54 b, 54 d withV-groove reference surfaces are secured to the substrate-holder 18 viadowel pins 58 and adhesive. The insert 54 c in the remaining corner ofthe substrate-holder 18 has a simple flat (horizontal) surface 60 and issecured to the substrate-holder 18 via a screw thread and adhesive.

With reference to FIG. 6B, the reference surfaces 55, 56, 57, and 60 onthe inserts 54 a, 54 b, 54 c, 54 d that are secured into thesubstrate-holder 18 are designed to rest in contact with datums 64disposed on the substrate-holder support 19. In the illustratedembodiment, the datums 64 include hemispherical surfaces and aredisposed at four corners on the substrate-holder support 19.

As before, when the reference surfaces 55, 56, 57, 60 of the inserts 54a, 54 b, 54 c and 54 d are received on the datums 64 of thesubstrate-holder support 19, the datums 64 and the reference surfaces55, 56, 57, and 60 locate the substrate-holder 18 in a selected positionin a selected plane. Further, if the substrate-holder 18 is removed fromthe substrate-holder support 19, and then re-seated on thesubstrate-holder support 19, the substrate-holder 18 will locate itselfin the identical plane and in the identical position within the plane.In addition, any substrate-holder 18 with the reference surfaces 55, 56,57, 60 set in the same positions and at the same heights (inserts 54 aand 54 c are adjustable for this purpose) will be accurately located inthe same plane and in the same position within the plane with respect tothe substrate-holder support 19.

Other embodiments for providing accurate and repeatable positioning ofthe substrate-holder 18 on a substrate-holder support 19 are includedwithin the scope of the present invention. Possible alternativeembodiments include, but are not limited to:

-   -   a) Providing three or more recesses in the bottom surface of the        substrate-holder 18 and an equal number of matching projections        in the top surface of the substrate holder support 19. All        recesses, when engaged with the matching projections on the        substrate-holder 18, provide bearing surfaces to define the        resting location of the substrate-holder 18 in the axis        perpendicular to the top surface of the substrate-holder 18. If        at least two of the recesses, when engaged in the matching        projections on the substrate-holder support 19, also provide        restraint to motion in the plane of the substrate-holder 18 (at        a minimum two of the engaged elements must provide restraint to        motion in two perpendicular axes parallel to the plane of the        substrate-holder 18), the substrate-holder 18 will be firmly        located in three dimensions.    -   b) The equivalent arrangement to a), wherein the recesses are        disposed in the substrate-holder support 19 and the projections        are disposed on the lower surface of the substrate-holder 18.    -   c) Providing precisely machined (e.g. milled or ground) bottom        surface regions on the substrate-holder 18 designed to engage        three or more support projections on the substrate-holder        support 19 (this defines the plane of the substrate-holder 18        and defines its position in the z-axis). Fiducial surfaces are        also provided on the substrate-holder support 19 to restrict        motion in the x and y axes, and the substrate-holder 18 is urged        by a spring, a magnet, a vacuum or other compliant or biasing        means against these surfaces.    -   d) Providing a precisely-milled or ground flat bottom on the        substrate-holder 18 designed to engage a precisely-milled or        ground flat surface on the substrate-holder support 19 (this        defines the plane of the substrate-holder and defines its        position in the z-axis). Fiducial surfaces 72 are also provided        on the substrate-holder support 19 to restrict motion in the x        and y axes and the substrate-holder 18 is urged by a spring 73,        a magnet, a vacuum or other compliant or biasing means against        these surfaces (FIG. 5E).    -   e) Providing three or more bearing surfaces on the bottom of the        substrate-holder support 19 designed to rest upon        precisely-milled or ground flat surface regions on the substrate        holder 18 (this defines the plane of the substrate-holder 18 and        defines its position in the z-axis). Fiducial surfaces on the        substrate-holder support 19 are also provided to restrict motion        in the x and y axes and the substrate-holder 18 is urged by a        spring, a magnet, a vacuum or other compliant or biasing means        against these surfaces.    -   f) Providing three or more grooved (e.g. V-groove) recesses with        parallel axes in the lower surface of the substrate holder 18 to        engage with matching point supports on projections disposed on        the substrate-holder support 19. A fiducial surface is also        provided on the substrate-holder support 19, perpendicular to        the axes of the grooves. The substrate-holder 18 is urged by a        spring, a magnet, a vacuum or other compliant or biasing means        against the fiducial surface.    -   g) The equivalent arrangement to f), wherein the recesses are in        the substrate-holder support 19 and the projections are on the        lower surface of the substrate holder 18.    -   h) Providing two or more elongated grooved (e.g. V-groove)        recesses with parallel axes in the lower surface of the        substrate holder 18 to engage with matching elongated supports        (e.g. bar supports) projecting from the substrate holder support        19. A fiducial surface on the substrate-holder support 19 is        also provided that is perpendicular to the axes of the grooves.        The substrate holder 18 is urged by a spring, a magnet, a vacuum        or other compliant or biasing means against the fiducial        surface.    -   i) The equivalent arrangement to h), wherein the recesses are in        the substrate-holder support 19 and the projections are on the        lower surface of the substrate holder 18.    -   j) Any of the mechanisms described in b)-i) above, further        including two or more through-holes in the substrate holder 18        that are designed to locate the substrate holder on matching        conical projections on the substrate-holder support 19 (similar        to the fluid-reservoir disposed on the fluid-reservoir support        illustrated in FIG. 8).    -   k) The arrangement described in j) above, further including a        compliant or biasing means (e.g. a spring, a magnet, an        electro-magnet, or a vacuum) to bias the substrate-holder 18        onto the side of one or more of the conical projections.

l) Providing two parallel V-grooves 71 and four spherically tippedprecision adjustment screws or datums 64. A fiducial surface 72 isprovided on the substrate-holder support 19 that is perpendicular to theaxes of the grooves 71. The substrate-holder 18 is urged by a spring 73,a magnet, a vacuum or other compliant or biasing means against thefiducial surface 72 (FIG. 5D).

-   -   m) Providing a substrate-holder 18 with inserts 54 a, 54 b, 54 c        and 54 d having opposed pairs of V-grooves for mating with        datums 64 (for example, spherical or conical) disposed on the        substrate-holder support 19 (FIG. 5C).

Referring again to FIG. 1, in another embodiment, the use ofsubstrate-holders 18 that can be repeatably and accurately loaded onto asubstrate-holder support 89 is combined with a substrate-holder storage70 for temporally storing a multiplicity of substrate-holders 18 and thesubstrates 16 located thereon. A conveyor system (not shown) forremoving substrate-holders 18 from the substrate-holder storage 70,loading the substrate-holders 18 onto the substrate-holder supports 89for deposition of fluids on the substrates 16, and then returning thesubstrate-holders 18 to the substrate-holder storage 70 may also beincluded. Alternatively, the motion system on which the substrate-holdersupport 89 is disposed may directly access the substrate-holder storage70 to remove a substrate-holder 18 from the substrate-holder storage 70or to place a substrate-holder 18 therein.

Various components of the microarrayer assembly 10 described above maybe combined together in alternative embodiments in accordance with theinvention. When combined, various benefits may be achieved.

For instance, in one embodiment, substrate-holders 18 may be readilyloaded into, and removed from, the deposition area (the area generallybeneath the printheads) of the microarrayer assembly 10 without loss ofpositional accuracy. In other words, all properly calibratedsubstrate-holders 18, when mounted on the substrate-holder support 89,will position the top surfaces 17 of the substrates 16 in asubstantially identical plane, as well as in a substantially identicalposition in the plane.

In another embodiment, the number of substrates 16 that may be processedby the microarrayer assembly 10 is limited only by the number ofsubstrates 16 on each substrate-holder 18 and the available number ofsubstrate-holders 18 in the substrate-holder storage 70. This benefit isderived in embodiments where the microarrayer can autonomously accessthe substrate-holder storage 70.

In another embodiment, the substrate-holders 18 may be removed from, andadded to, the substrate-holder storage 70 while depositions are underwayon an active substrate-holder 18 loaded in the deposition area of themicroarrayer assembly 10. Therefore, it is not necessary to ceasespotting operations to load and unload substrates 16 orsubstrate-holders 18, as in existing microarrayers. It will beappreciated that deposition operations may continue indefinitely if,periodically, fresh substrates 16 are introduced into thesubstrate-holder storage 70 and processed substrates 16 are removed fromthe storage 70.

In another embodiment where the number of substrates 16 that can beprocessed is limited only by the capacity of the substrate-holderstorage 70 and not the size of the substrate-holder 18, relatively smallsubstrate-holders 18, holding, for example six to twenty glass-slidesubstrates 16 may be used, minimizing the size of the deposition areaand volume of the microarrayer assembly 10. Moreover, the use of smallsubstrate-holders 18 may negate the need for large, slow, overly complexand expensive motion elements that are required for larger mobilesubstrate-holders 18.

In yet another embodiment including a substrate-holder storage 70,manual loading/unloading of substrates 16 from the section of themicroarrayer 10 dedicated to deposition is eliminated. Automatic loadingand unloading of substrates 16 minimizes or eliminates sources of errorresulting from frequent human access to the deposition area.

In another embodiment that includes automatic loading of substrates 16into the microarrayer assembly 10, the area for droplet depositions (thedeposition chamber) is relatively closed and relatively undisturbed byhuman access. Therefore, well-controlled and stable environmentalconditioning of this area is possible. In another embodiment, separateenvironmental controls may be applied to the substrate-holder storage 70and the deposition area.

In yet another embodiment where relatively small substrate-holders 18are used, the exposure of the substrates 16 to the environment of thedeposition area can be relatively short. This may be of benefit, forexample, if the fluids being deposited are best kept cold, but may be ata higher temperature for deposition. In yet another embodiment, themicroarrayer assembly 10 may be scaled in size, since the size andfunctions of the deposition equipment is not tied to the number ofsubstrates 16 being processed. Therefore, as later described, arrayerdesigns can be realized using multiple deposition engines workingcooperatively to significantly increase throughput.

c) Fluid Reservoirs and Fluid Reservoir Holders

A variety of fluid reservoirs 20 may be used to supply the fluid samplesto the printheads 12 of the microarrayer assembly 10. In one embodiment,a microplate 20 with 96 wells, or a multiple of 96 wells is used. Theuse of the higher density microplates 20, for example, having 1536wells, is suited to solid-pin deposit element 14 implementations sincevery narrow pin tips 26 are readily fabricated with solid pins 14.

In microarrayer assembly 10 embodiments that include a high densityfluid-reservoir array, such as microplates 20 with 1536, 3456 or 6144wells, greater positional accuracy is required to hold the fluidreservoir 20 in the microarrayer assembly 10. With reference to FIG. 7,a fluid-reservoir holder 21 that includes inserts 76, 78, 80, and 82designed to engage datums 65 disposed on a fluid-reservoir holdersupport 22 is illustrated. Similar to the mounting arrangement for thesubstrate-holder 18 and the substrate-holder support 19 describedearlier in FIGS. 5B and 6B, the inserts and the datums respectivelydisposed on the fluid reservoir holder 21 and the fluid reservoir holdersupport 22 enable fluid-reservoirs 20 to be accurately and repeatablyloaded onto into the microarrayer assembly 10. In another embodiment,three inserts are provided to engage three datums as described withreference to FIGS. 5A and 5B. In other embodiments, any of thereferencing or planarization systems described previously for use withthe substrate-holder 18 and the substrate-holder support 19 can be usedwith either a fluid reservoir 20 mounted directly onto a fluid-reservoirholder support 22 or a fluid reservoir holder 21 mounted on a fluidreservoir-holder support 22, the fluid-reservoir holder holding thefluid-reservoir 20. In addition, a fluid-reservoir holder storage 83 maybe included in the microarrayer assembly 10. Similar to the substratehandling mechanisms described earlier, the fluid reservoirs holders 21may be robotically removed from the fluid-reservoir holder storage 83and placed back into the storage 83 after use. This provides many of thesame advantages described earlier with respect to the automated handlingof substrates.

With continued reference to FIG. 7, the fluid reservoir 20 is referencedagainst precisely machined raised edges 84 disposed on thefluid-reservoir holder 21. Resilient spring clips 86 are then used tohold the fluid reservoir 20 firmly against the raised reference edges 84of the fluid-reservoir holder 21.

In one embodiment, overall fluid-reservoir holder and fluid-reservoirholder support system position accuracy is within ±0.02″ in the x, y,and z-axes. In a preferred embodiment, overall fluid-reservoir holderand fluid-reservoir holder support system position accuracy is within±0.002″ in the z-axis and within ±0.01″ in the x and y axes. In a morepreferred embodiment, overall fluid-reservoir holder and fluid-reservoirholder support system position accuracy is within ±0.0002″ in the z-axisand within ±0.001″ in the x and y axes.

With reference to FIG. 8, an alternative embodiment of a fluid-reservoirholder 121 is illustrated. The fluid-reservoir holder support 122includes a precisely fabricated flat top surface 124 designed tointerface with a precisely fabricated flat undersurface 126 of thefluid-reservoir holder 121. Precisely machined through holes 128 in thefluid-reservoir holder 121 are configured to accept reference pins ordatums 130 with conical surface segments. The fluid-reservoir 20, wheninstalled, is referenced against raised edges 184 disposed on thefluid-reservoir holder 121. Resilient spring clips 186 may be used tohold the fluid-reservoir 120 firmly against the raised reference edges184. Two pairs of magnetic elements 129, one disposed on the lowersurface of the fluid-reservoir holder 121 and one disposed on thefluid-reservoir holder support 122 provide a pre-load to hold the lowersurface of the fluid-reservoir holder 121 firmly against thefluid-reservoir holder support 122 and also to bias the side of theholes 128 against the vertical elements of the reference pins 130 toconstrain any motion in the horizontal plane.

In some embodiments, especially when using fluid-reservoirs 20 thatinclude a dense array of wells, it is desirable to place lids on thefluid-reservoirs 20 when they are not in use to minimize evaporation ofthe fluid and the introduction of airborne contaminants or particulatesinto the fluid. In such cases an automated de-lidding station can beadded to the microarrayer assembly 10 to remove the lid before thefluid-reservoir 20 is used to supply fluids to the deposit elements 14,and to replace the lid after the completion of use of thefluid-reservoir 20.

d) Microarrayer Architectures

The deposition of micro fluid droplets in ordered arrays upon substrates16 requires a minimum set of physical motions to bring the printhead 12into proximity with all fluid retention locations of the fluid reservoir20 and all deposition sites on the substrate 16. Precision linear orrotational motion systems that are computer controlled and, in someinstances, have precision positional feedback, are assumed to beincluded in the following embodiments. The physical, electrical andcomputer program elements required to realize such precision motioncontrol, with positioning capability in the micron or sub-micron range,are well known to those skilled in the art, and are therefore notdescribed further.

In various embodiments, any of the assemblies described may beconfigured with covers, heaters, chillers, humidifiers, dehumidifiers,control systems and other elements to provide a controlled environmentin which the fluid droplets are deposited upon the substrates 16. Insome cases, it may be preferable to provide temperature control to theentire microarrayer assembly 10, and in some cases individual elementsof the microarrayer assembly 10 may be controlled e.g. localized coolingof the fluid reservoir 20 to inhibit denaturing of sensitive biologicalsamples. Air filtering to inhibit contamination of the fluid samples orthe substrates 16 by airborne particulates can also be provided.Similarly, the substrate-holder storage 70 and the fluid-reservoirholder storage 83 can be similarly environmentally conditioned, with thesame, or with different environmental parameters.

i) Microarrayer Architectures for “Equal Exposure Time” Spotting

In various embodiments, the motion control system, in addition tocontrolling the relative positions of the substrate-holders 19 and thefluid-reservoir holders 21, is designed and arranged to:

-   -   i) move the printhead 12 and/or the fluid reservoir 20,        relatively, to dip the deposit elements 14 into the fluid        reservoir 20 to capture fluid;    -   ii) move the printhead 12 and/or the substrate 16, relatively,        to position the desired fluid deposition location on the        substrate 16 under the printhead 12;    -   iii) move the printhead 12 and/or the substrate 16, relatively,        so that the deposit element 14, or the fluid droplet on the tip        of the deposit element 14, contacts the top surface 17 of the        substrate 16; and,    -   iv) vary the speed of motion of the various moving elements, or        equivalently, introduce variable delays in the motions,        calculated and applied such that for every deposited droplet of        fluid on the substrate 16 or substrates 16, the fluid captured        by the deposition element 14 is exposed to the surrounding        atmosphere for substantially the same amount of time between its        extraction from the fluid reservoir 20 and its deposition on the        substrate 16 nomatter from which part of the fluid reservoir 20        the fluid is extracted, nor where on the substrate 16, or on        which substrate 16 the fluid droplet is deposited.

The use of the motion control system in this manner equalizes theevaporation of the fluid being carried by the deposit element 14 duringthe time period between fluid capture from any fluid reservoir 20location, to deposition on any droplet deposition site on the substrate16. The arrangement is conceptually illustrated in FIG. 9. Referring toFIG. 9, substrates 16 are secured to the substrate-holder 18 and a fluidreservoir 20 is secured to a fluid reservoir holder 21. Thesubstrate-holder 18 includes one of the planarization systems earlierdescribed to accurately and repeatably load the substrate-holder 18 ondatums 64 disposed on the substrate-holder support 19. Likewise, thefluid reservoir holder 21 which holds the fluid reservoir 20 includesone of the planarization systems earlier described to accurately andrepeatably load the fluid-reservoir holder 21 on datums 65 disposed onthe fluid-reservoir-holder support 22. In the illustrated embodiment,the substrate-holder support 19 and the fluid reservoir holder support22 are a single, integrally formed, platen 89. For illustrationpurposes, a single deposit element 14 is held by a printhead 12,however, the printhead 12 may hold a plurality of deposit elements 14.Fluid is captured from the fluid-reservoir 20 by dipping the tip 26 ofthe deposit element 14 into the wells. Fluid is then spotted on thesubstrate 16 by touching the tip 26 of the deposit element 14, or thefluid droplet on the tip of the deposit element 14, onto the desiredposition of the substrate 16. A motion control system, for instance, acomputer 90, provides stimuli to actuators to move the platen 89 and theprinthead 12, such that the time of exposure of the fluid droplet on thetip 26 of the deposit element 14 to the air is the same for the shortestpath and the longest path between a well and a deposition location, andall paths in between. In FIG. 9, the shortest path is represented bypath “A” and the longest path is represented by path “B”. Equalizationof the exposure times as described may be realized by extending theexposure times for all depositions to match the longest exposure time,for example, the time taken for the deposit element 14 and the platen 89to move relatively over path “B”. The longest exposure time may becalculated based on the motion parameters of the mobile elementsinvolved, or by measurement of the exposure time associated with thelongest path length involved, i.e. path “B”, at the maximum operatingacceleration, maximum velocity, and maximum deceleration. The extensionof the exposure times for paths shorter than path B, (for instance, pathA), may be applied as delays between the various motions (e.g. delay inlowering the printhead 12 to deposit the droplet on the substrate 16 orby slowing the speed of one or more of the printhead 12 and the platen89).

ii) Microarrayer Architectures With Combined Substrate Motion and FluidReservoir Motion

Referring again to FIG. 1, one embodiment of an architecture for themicroarrayer assembly 10 is further described. In the illustratedembodiment, the substrate-holder 18 and the fluid-reservoir holder 21are shown mounted on the shared platen 89 which is movable in an X-Yplane when disposed beneath the printhead 12. The shared platen 89 maybe operated to access both the substrate-holder storage 70 and the fluidreservoir holder storage 83. The substrate-holder storage or “hotel” 70includes a vertically mobile rack of vertically-separated receptaclespaces into which substrate-holders 18, along with the substrates 16mounted thereon, may be initially manually or automatically installed.Each substrate-holder 18 is supported in the substrate-holder storage 70by rails 92 which extend slightly under the substrate-holder 18 onopposite sides. Each receptacle is spaced apart, or can be moved apart,by a distance that will permit access between the receptacle spaces bythe platen 89 of the microarrayer assembly 10. In one embodiment,substrate-holders 18 are transferred to the platen 89 of themicroarrayer assembly 10 by:

-   -   a) vertically moving the rack of substrate-holders 18 in the        substrate-holder storage 70 to position a space below the        substrate-holder 18 to be removed in the plane of motion of the        platen 89. The space must be large enough for entry of the        platen 89,    -   b) moving the platen 89 beneath the substrate-holder 18 to be        transferred,    -   c) vertically moving the substrate-holders 18 in the        substrate-holder storage 70 to lower the desired        substrate-holder 18 onto the platen 89 such that the inserts 54        of the substrate-holder 18 contact and engage the datums 64 on        the platen 89 to accurately position the substrate-holder 18 on        the platen 89 in a defined plane as earlier described. At this        time, the substrate-holder 18 is no longer supported by the        rails 92, and    -   d) moving the platen 89 in the y dimension to withdraw the        substrate-holder 18 from the substrate-holder storage 70.

Returning a substrate-holder 18 to the substrate-holder storage 70 maybe effected by the same series of steps in reverse. An equivalent set ofsteps may be used to load and unload fluid-reservoir holders 21 from thefluid-reservoir holder storage 83.

iii) Microarrayer Architectures With Separate Substrate Motion and FluidReservoir Motion

With reference to FIG. 10, another embodiment of a microarrayer assembly200 is illustrated. In this embodiment, the motion of a printhead 212including one or more deposit elements 214 is coordinated with themotions of a substrate-holder support 219 and a fluid reservoir holdersupport 222 which are actuatable in separate X-Y planes. Thesubstrate-holder support 219 and the fluid reservoir holder support 222are independently mobile, but move in a coordinated manner to effect thedeposition of fluid droplets upon substrates 216. The substrate-holder218 and the fluid reservoir holder 221 are respectively held on thesubstrate-holder support 219 and on the fluid reservoir holder support222 using the planar referencing systems described earlier. In anotherembodiment, a fluid reservoir 220 is directly positioned on the fluidreservoir holder support 222 using the planar referencing systemsdescribed earlier without the use of a holder 221.

With continued reference to FIG. 10, the fluid-reservoir holder support222, in one embodiment, may:

-   -   a) move such that any desired location of the fluid reservoir        220 is positioned directly below the deposit elements 214 of the        print head 212 to allow charging or re-charging of those deposit        elements 214 by having the deposit elements 214 dip into the        fluids held in the fluid reservoir 220.    -   b) move clear of the vertical path of the print head 212        assembly to allow the print head 212 to descend below the X-Y        plane in which the fluid reservoir 220 moves when disposed        beneath the printhead to i) deposit droplets of fluid onto the        substrates 216 or ii) access a wash station 224, which is        vertically below the printhead 212.    -   c) move clear of the vertical path of the printhead 212 into an        area in which the fluid-reservoir holder 221 can be accessed for        manual or robotic replacement of the fluid-reservoir holder 221,        for example, to a storage 270.

Similarly, the substrate-holder support 219 may move in a planedisplaced from the plane of motion of the fluid-reservoir holder support222 to:

-   -   a) position the substrates 216 such that any desired respective        set of printing locations on any substrate 216 is directly below        the deposit elements 214 of the printhead 212 to allow        deposition of a fluid sample, or fluid samples, on the substrate        216 when the printhead 212 is lowered such that the tip 226 of        each deposit elements 214 is in contact with, or the droplet of        fluid on the tip 226 of each deposit elements 214 is in contact        with, the top surface 217 of the substrate 216,    -   b) move clear of the vertical path of the printhead 212 to        enable the printhead 212 to descend unobstructed below the x-y        plane in which the substrate-holder support 219 moves when it is        disposed beneath the printhead 212 to access components or        equipment below such as the wash station 224, and    -   c) move clear of the path of the printhead 212 into an area from        which the substrate-holder 218 may be accessed for its manual or        robotic removal or replacement for the purpose of removal or        replacement of the substrates 216, for example, to the storage        270.    -   d) Position sensing (e.g. using position encoders) allows        automated monitoring of the location of the substrate-holder        support 219, the fluid-reservoir holder support 222, and the        printhead 212. Computer control inhibits any motions of these        components that would result in unintentional contact between        them.

Deposition of droplets of biological or chemical liquid material uponthe substrates 216 may be achieved in one embodiment by: (note: thisprocedure assumes that the deposit elements 214 for example, solid pins214, are clean and that the fluid reservoir(s) 220 and the substrates216 are already supported on the fluid reservoir holder support 222 andthe substrate-holder support 219 respectively)

-   -   a) moving the printhead 212 to a fully raised position in a        Z-axis,    -   b) moving the fluid-reservoir holder support 222 in an X-Y plane        to align a first set of wells of the fluid-reservoir 220 under        the deposit elements 214 of the printhead 212 while        simultaneously moving the substrate-holder support 219 in tandem        with the fluid-reservoir holder support 222 such that the        absolute distance between the fluid-reservoir 220 and the        substrates 216 remains substantially constant,    -   c) moving (lowering) the printhead 212 in the Z-axis such that        the tips 226 of the deposit elements 214 are immersed in the        fluid samples and small amounts of fluid are captured on the        tips of the deposit elements 214 when the deposit elements 214        are subsequently lifted out of the liquid samples,    -   d) moving (raising) the printhead 212 away from the        fluid-reservoir 220 a sufficient distance that the        fluid-reservoir 220 can be moved in its X-Y plane without        contacting the deposit elements 214 or any part of the printhead        212,    -   e) moving the fluid-reservoir 220, in its X-Y plane, away from        the vertical path of the printhead 212 towards its starting        position to allow the printhead 212 to descend below the X-Y        plane of the fluid-reservoir 220,    -   f) moving the substrate-holder support 219 to align a first set        of desired print locations on one substrate 216 under the        printhead 212,    -   g) lowering the printhead 212 in the Z-axis such that the tip        226 of each deposit element 214 is in contact with, or the        droplet of fluid on the tip 226 of each deposit element 220 is        in contact with, the top surface 217 of the substrate 216,        thereby depositing small droplets of fluid (one droplet per pin)        onto the top surface 217 of the substrate 216,    -   h) raising the printhead 212 above the X-Y planes of both the        fluid-reservoir-holder support 222 and the substrate-holder        support 219 such that either can move in their respective planes        without contacting the deposit elements 214 or any part of the        printhead 212,    -   i) moving the substrate-holder support 219 away from the        vertical path of the printhead 212,    -   j) repeating steps b) to i) for the next set of desired        deposition sites on the same substrate 216 using the first set        of wells on the fluid-reservoirs 220 until all desired        deposition sites on that substrate 216 have been spotted with        the fluid from that set of wells (note: users may wish to spot        the same sample several times on a substrate 216 so that they        can assess repeatability),    -   k) repeating steps b) to i) for desired deposition sites on the        second substrate 216 and on all other desired substrates 216 on        the substrate-holder 218 using the same set of wells until all        desired deposition sites on all substrates 216 have been spotted        with the fluid from the first set of wells of the fluid        reservoir 220,    -   l) once all desired deposition sites on all substrates 216 on        the substrate-holder 218 have been spotted with the fluid        samples from the first set of wells on the fluid-reservoir 220,        moving both the fluid-reservoir holder support 222 and the        substrate-holder support 219 aside in their respective X-Y        planes to allow the printhead 212 to descend to a wash station        224 where the deposit elements 214 are washed and dried to avoid        carryover of fluid samples to the next set of printed spots, and    -   m) repeating sequence a) through l), but now for the next set of        fluid samples (i.e. the next set of wells in the fluid-reservoir        220) and then for all sets of fluid samples until all desired        samples (perhaps from multiple fluid-reservoirs 220) have been        spotted on all desired deposition sites on all substrates 216.

In another embodiment, it may be desirable to add wash-and-dry cycles tothe spotting sequence described above, after a certain number ofdroplets have been deposited to avoid evaporative sample build-up on thedeposit elements 214. These intermediate wash-and-dry cycles have beenignored in the above description to avoid complicating the narration ofthe deposition procedure.

In FIG. 10, the fluid reservoir holder support 222 moves, when disposedbeneath the printhead 212, in an x-y plane above the x-y plane of motionof the substrate-holder support 219. In another embodiment, thesubstrate-holder support 219 may move in a plane of motion that is abovethe plane of motion of the fluid-reservoir holder support 222. In thisarrangement, the fluid-reservoir holder support 222 may be heldstationary beneath the printhead 212 throughout the printing cycle for agiven set of wells of the fluid-reservoir 220.

In one embodiment, the procedure to deposit fluid on a substrate 216using the alternative setup just described is as follows. As a firststep, the desired first set of wells of the fluid-reservoir 220 arepositioned below the deposit elements 214 of the printhead 212. The tips226 of the deposit elements 214 are then immersed in the fluid samplesby lowering the printhead 212. As a further step, the printhead 212 israised before the substrate-holder support 219 travels to position adesired set of print locations below the printhead 212. The printhead212 is then lowered to deposit fluid on the substrate 216 and thenraised before the substrate-holder support 219 is moved away from thevertical path of the printhead 212. In the next step, the printhead 212is lowered again into the same wells of the fluid-reservoir 220. Thisprocess is continued until all desired deposition locations have beenspotted from the first set of wells on the fluid-reservoir 220. Aftermoving the substrate-holder support 219 and fluid-reservoir holdersupport 222 aside to wash the deposit elements 214, a new set of wellsare located under the deposit elements 214 of the printhead 212. Thisprocess is repeated until all desired fluid samples have been depositedat all desired locations on all desired substrates 216.

In other embodiments, it will be appreciated that a storage 270 and ameans for transferring the substrate-holders 218 and the fluid-reservoirholders 221 between the storage 270 and the substrate-holder support 219and the fluid-reservoir holder support 222 may be provided. The transferof substrate-holders 219 and fluid-reservoir holders 221 to and from thestorage 270 and their respective mobile supports 219, 222 can beimplemented in the same manner earlier described.

The various architectures described may be used with solid pins 214since the architectures can be used to minimize the cycle time for thecontinuous dip-and-deposit cycle that is required when using solid pins214. Furthermore, in the described architectures, since the depositelements 214 make the same vertical motion cycle for every deposition(i.e. the vertical path of the tip 226 of any deposit element 214 is thesame for any deposition), the architectures are suitable for spottingfluids using the “equal exposure time” method with relatively littleimpact on overall deposition rates. Moreover, since the printhead 212does not move laterally, there is no differential drying effect on oneside of the pin 214 as would be experienced from “windage” effects inembodiments where the printhead is moved in an X-Y plane. Differentialdrying on one side of the pin 214 can affect the shape of the depositeddroplet. Using the architectures described, drying of the sample on thepin 214 will occur uniformly around the pin 214 since the printhead 212is only moved vertically.

iv) Microarrayer Architectures with a Plurality of Printheads

The separation of planar motions of the substrates holder support andthe fluid reservoir support, combined with motion of the printhead in anaxis perpendicular to the supports, permits further architectures to bedeveloped that significantly increase deposition rates. The resultingarchitectures are particularly beneficial for increasing the depositionrates that may be achieved using solid pins. However, other depositionelements such as quill pins, pens and ink jet devices can also beeffectively used.

With reference to FIG. 11, a microarrayer assembly 310 having aplurality of printheads 312 is illustrated. In the illustratedembodiment, the motion of two independently mobile printheads 312 a, 312b (collectively 312), each including one or more deposit elements 314,for instance solid pins 314, and each constrained to move in a verticalaxis (the Z-axis), are coordinated with the motions of a) asubstrate-holder support 319, which may move in any direction within anx-y plane substantially perpendicular to the aforementioned verticalaxis when disposed beneath the printheads, and b) a fluid-reservoirholder support 322, which may move in any direction within an x-y planeseparate from the plane of motion of the substrate-holder support 319when disposed beneath the printheads, also essentially perpendicular tothe aforementioned vertical axis. The axes of motion of the printheads312 a and 312 b are parallel and displaced from each other laterally(horizontally). The substrate-holder support 319 and fluid-reservoirholder support 322 are independently mobile, but move in a coordinatedmanner to effect the deposition of fluid droplets upon the substrates316. The substrates 316 and the fluid reservoirs 320 may be respectivelymounted on a substrate-holder 318 and a fluid-reservoir holder 321 asearlier described. The required motorized linear stages associated withthe X, Y and Z motions have been omitted from the drawing for clarity.

With continued reference to FIG. 11, the stages holding the twoprintheads 312 are mounted at the same height, and are separated in theY dimension by a distance larger than the width of the substrate-holder318 (or its support holder 319 if larger). The deposit elements 314 aremounted in the printheads 312 with a repetitive spacing which is afunction of the well spacing of the fluid-reservoirs 320.

The fluid reservoir 320 (such as a microplate, with 96 wells, or amultiple of 96 wells), for supplying fluid samples to the tips of thedeposit elements 314 of the printheads 312 is mounted on afluid-reservoir holder 321, which in turn is held on the roboticallycontrolled substrate-holder support 322 using the planarization systemdescribed earlier. In one embodiment, the fluid-reservoir-holder support322 may:

-   -   a) move such that any set of wells of the fluid-reservoir 320        are positioned directly below the deposit elements 314 of either        of the printheads 312 to allow charging or re-charging of those        deposit elements 314 by having the deposit elements 314 dip into        the fluids held in the wells,    -   b) move clear of the vertical path of the printheads 312 to        allow the printheads 312 to descend below the X-Y plane in which        the fluid-reservoir 320 moves when disposed beneath the        printhead to i) deposit spots of fluid onto the substrate 316,        or ii) access wash stations 324 below (in the illustrated        embodiment, there are separate wash stations 324 for each        printhead 312, although in another embodiment a single mobile        wash station is used), and    -   c) move clear of the vertical paths of both printheads 312 into        an area in which the fluid-reservoir holders 321 can be accessed        for manual or robotic replacement of the fluid-reservoir holders        321 (and hence the fluid reservoirs 320).

The substrate-holder support 319 is mechanically arranged such that, inone embodiment, it may:

-   -   a) move such that any desired respective set of printing        locations on any substrate 316 can be positioned directly below        the deposit elements 314 on either printhead 312 to allow        deposition of a fluid sample, or fluid samples, on the substrate        316 when the printheads 312 are lowered such that the tip 326 of        each deposit elements 314 is in contact with, or the droplet of        fluid on the tip 326 of each deposit element 314 is in contact        with, the top surface 317 of the substrate 316,    -   b) move clear of the vertical path of the printheads 312 to        allow the printheads 312 to descend below the X-Y plane in which        the substrate-holder support 319 moves when disposed beneath the        printheads to access a wash station 324, or wash stations 324,        below, and    -   c) move clear of the path of the printheads 312 into an area        from which the substrate-holder 318 may be accessed for manual        or robotic removal or replacement of the substrate-holder 318        for the purpose of removal or replacement of the substrates 316.

Deposition of droplets of biological or chemical fluid material upon thesubstrate(s) 316 may be achieved with two printheads 312, for example,using either of two methods: sequential or concurrent washing. Themethod associated with sequential washing is described first (note: thisprocedure assumes that the deposit elements 314 of both printheads 312have been washed and that fluid-reservoirs 320 and substrates 316 arealready installed in the microarrayer assembly 310). Droplet depositionusing a sequential washing cycle may be achieved by the followingsequence of actions; however, other motion sequences may also be used,and the following is by way of example only:

a) move printheads 312 a, 312 b to their fully raised positions,

-   -   b) move the fluid-reservoir holder support 322 in the X-Y plane        to align a first set of wells of the fluid-reservoir 320 under        the deposit elements 314 of the printhead 312 a while        simultaneously moving the substrate-holder support 319 in tandem        with the fluid-reservoir holder support 322 such that the        absolute distance between the fluid-reservoir holder support 322        and the substrate-holder support 319 remains substantially        constant,    -   c) move the substrate-holder support 319 to align a first set of        desired print locations on a first substrate 316 under the        deposit elements 314 of the printhead 312 a,    -   d) lower the printhead 312 a in the Z-axis such that the tips        326 of the deposit elements 314 of the printhead 312 a are        immersed in the first set of fluid samples, and a small amount        of fluid is captured on the tips 326 of the deposit elements 314        when the deposit elements 314 are subsequently lifted out of the        wells,    -   e) raise the printhead 312 a away from the fluid-reservoir 320 a        sufficient distance such that the fluid-reservoir holder support        322 can be moved in its X-Y plane without contacting the deposit        elements 314 or the printheads 312,    -   f) move the fluid-reservoir holder support 322 away from the        vertical axis of the printhead 312 a to enable the printhead 312        a to descend unobstructed below the X-Y plane of motion of the        fluid-reservoir holder support 322,    -   g) lower the printhead 312 a in the Z-axis such that the tip 326        of each deposit element 314 is in contact with, or the droplet        of fluid on the tip 326 of each deposit element 314 is in        contact with, the top surface 317 of the substrate 316, thereby        depositing small droplets of fluid (one droplet per deposit        element 314) onto the top surface 317 of the substrate 316,    -   h) raise the printhead 312 a above the X-Y planes of motion of        both the fluid-reservoir holder support 322 and the        substrate-holder support 319 such that either can move in their        respective planes without contacting the deposit elements 314 or        the printheads 312,    -   i) repeat steps b) through h) for the second and subsequent sets        of desired print locations on the first substrate 316 using the        first set of wells of the fluid-reservoir 320,    -   j) when all desired print locations on the first substrate 316        have been spotted using the first set of wells of the        fluid-reservoir 320, repeat b) through h) for the next and        subsequent substrates 316 with the first set of wells of the        fluid-reservoir 320,    -   k) at the completion of spotting of all substrates 316 with the        first set of wells of the fluid-reservoir 320, move the        fluid-reservoir holder support 322 in its X-Y plane of motion to        align a second set of wells of the fluid-reservoir 320 under the        deposit elements 314 of the printhead 312 b while simultaneously        moving the substrate-holder support 319 in tandem with the        fluid-reservoir holder support 322 such that the absolute        distance between the fluid-reservoir holder support 322 and the        substrate-holder support 319 remains substantially constant,    -   l) repeat steps c) through j), but now using the printhead 312        b. While the printhead 312 b is spotting, the deposit elements        314 of the printhead 312 a are washed. After washing, the        printhead 312 a is raised to its fully raised position above the        X-Y planes of motion of the substrate-holder support 319 and the        fluid-reservoir holder support 322, and the printhead 312 a        waits unused until the printhead 312 b enters a wash cycle, and    -   m) continue repeating the above described sequence, changing        operating printheads 312 at each wash cycle event, until all        desired print locations are spotted on all substrates 316 from        all desired sets of wells in the fluid-reservoir(s) 320.

Alternatively, deposition of droplets of biological or chemical fluidmaterial upon the substrate(s) 316 can be achieved with the twoprintheads 312 a, 312 b using the concurrent washing method. Withcontinued reference to FIG. 11, one example of the concurrent washingmethod is as follows:

-   -   a) move the printheads 312 a, 312 b to their fully raised        positions as shown in FIG. 11,    -   b) move the substrate-holder support 319 under the printhead 312        b,    -   c) move the fluid-reservoir holder support 322 in its X-Y plane        of motion to align a first set of wells of the fluid-reservoir        320 under the deposit elements 314 of the printhead 312 a,    -   d) move the substrate-holder support 319 to align a first set of        deposition locations on the first substrate 316 for the first        set of fluid samples under the deposit elements of the printhead        312 a,    -   e) lower the printhead 312 a in the Z-axis such that the tips        326 of the deposit elements 314 of the printhead 312 a are        immersed in the first set of fluid samples, and a small amount        of fluid is captured on the tips 326 of the deposit elements 314        when the deposit elements 314 are subsequently lifted out of the        liquid samples,    -   f) raise the printhead 312 a away from the fluid-reservoir        holder support 322 a sufficient distance that the        fluid-reservoir 320 can be moved in its X-Y plane of motion        without contacting the deposit elements 314 or the printhead 312        a,    -   g) move the fluid-reservoir holder support 322 in it's X-Y plane        of motion to align a second, new set of wells of the        fluid-reservoir 320 under the pins 314 of the printhead 312 b.        The separation of the printheads 312 a, 312 b is such that with        any set of wells of the fluid-reservoir(s) 320 located under the        deposit elements 314 of the printhead 312 b, the printhead 312 a        can descend unobstructed below the X-Y plane of motion of the        fluid-reservoir holder support 322,    -   h) lower both printheads 312 a, 312 b in the Z-axis such        that (i) the tips 326 of the deposit elements 314 of the        printhead 312 a are in contact with, or the droplet of fluid on        the tip 326 of each deposit element 314 of the printhead 312 a        is in contact with, the top surface 317 of the substrate 316,        thereby depositing small droplets of fluid (one droplet per        deposit element 314) onto the top surface 317 of the substrate        316, and (ii) the tips 326 of the deposit elements 314 of the        printhead 312 b are immersed in the second set of fluid samples,        and are charged with fluid samples,    -   i) raise both printheads 312 a, 312 b above the X-Y planes of        motion of both the fluid-reservoir holder support 322 and the        substrate-holder support 319 such that the fluid-reservoir        holder support 322 and the substrate-holder support 319 can move        in their respective planes without impacting the printheads 312        a, 312 b,    -   j) move the substrate-holder support 319 such that the first set        of desired deposition locations on the first substrate 316 for        the second set of fluid samples is directly below the deposit        elements 314 of the printhead 312 b,    -   k) move the fluid-reservoir holder support 322 such that the        first set of wells of the fluid-reservoir 320 is again directly        below the deposit elements 314 of the printhead 312 a,    -   l) lower both printheads 312 a, 312 b in the Z-axis such        that (i) the tips 326 of the deposit elements 314 of the        printhead 312 b are in contact with, or the droplet of fluid on        the tip 326 of each deposit element 314 is in contact with, the        top surface 317 of the substrate 316, thereby depositing small        droplets of fluid (one droplet per deposit element 314) onto the        top surface 317 of the substrate 316, and (ii) the tips 326 of        the deposit elements 314 of the printhead 312 a are immersed        again in the first set of wells of the fluid-reservoir 320 and        are recharged with fluid samples,    -   m) raise both printheads 312 a, 312 b above the X-Y planes of        motion of both the fluid-reservoir holder support 322 and the        substrate-holder support 319 such that both the fluid-reservoir        holder support 322 and the substrate-holder support 319 can move        in their respective planes without impacting the printheads 312        a, 312 b,    -   n) move the substrate holder support 319 to align a second set        of deposition locations on the first substrate 316 for the first        set of fluid samples under the deposit elements 314 of the        printhead 312 a,    -   o) move the fluid-reservoir holder support 322 in it's X-Y plane        of motion to align the second set of wells of the        fluid-reservoir 322 under the deposit elements 314 of the        printhead 312 b,    -   p) repeat steps h) through o), but now for the second, and all        other desired deposition locations, until all desired deposition        locations on the first substrate 316 have received depositions        from the first two sets of wells of the fluid-reservoir 320,    -   q) repeat the above sequence until all desired substrates 316 on        the substrate-holder 318 have been spotted from the first two        sets of wells of the fluid-reservoir 320,    -   r) move the fluid-reservoir holder support 322 and the        substrate-holder support 319 away from the axes of motion of        both the printheads 312 a, 312 b and lower the printheads 312 a,        312 b to the wash stations 324 below,    -   s) after washing the deposit elements 314 of both printheads 312        a, 312 b concurrently, restart at step a) but with the use of        the third and fourth set of wells on the fluid-reservoir 320,        and    -   t) repeat the above sequence for each subsequent set of wells on        the fluid-reservoir 320 until all desired deposition locations        on all substrates 316 on the substrate-holder 318 have been        spotted from all wells of the fluid-reservoir 320.

Although the microarrayer assembly 310 illustrated in FIG. 11 includesthe fluid-reservoir holder support 322 moving in an X-Y plane of motionabove the plane of motion of the substrate-holder support 319, inanother embodiment, the substrate-holder support 319 moves in an X-Yplane of motion that is above the plane of motion of the fluid-reservoirholder support 322. Further embodiments may include any of themicroarrayer assembly components earlier described, for instance, asubstrate-holder storage and a fluid-reservoir holder storage. Thedescribed printing methods can also be altered to provide equal exposuretime printing.

Notwithstanding the suitability of the architecture shown in FIG. 11 foruse with solid deposition pins 314, the architecture can also be usedwith quill pins, pens or ink jet devices. One beneficial aspect of thearchitecture of FIG. 11 using these devices is the potentialminimization of, or elimination of, spotting time lost because ofwashing. Wash times for quill pins, pens and ink-jet devices can belonger than that for solid pins 314 because of the difficulty influshing sample fluids from inner, less-accessible surfaces. Using asequential wash method and the architecture of FIG. 11, deposition ratesusing quill pins, pens, or aspirating ink-jet dispensers may beincreased substantially since one printhead 312 may continue depositionoperations while the deposit elements 314 on the other printhead 312 arebeing washed.

With reference to FIG. 12, in another embodiment, the deposition rate ofa microarrayer assembly 410 may be increased by using four independentlymobile printheads 412. One advantage of this embodiment is that thedeposition rate benefit derived from the interlacing of dip-and-depositactions of two printheads as described with respect to FIG. 11 can beachieved without losing any deposition time due to a wash cycle. In theillustrated embodiment, the motion of the four independently mobileprintheads 412, each constrained to move in a vertical axis, arecoordinated with the motions of a) a substrate-holder support 419, whichmay move in any direction within a plane substantially perpendicular tothe aforementioned vertical axis when it is disposed beneath theprintheads, and b) a fluid-reservoir holder support 422, which may movein any direction within a separate plane when it is disposed beneath theprintheads, also essentially perpendicular to the aforementionedvertical axis, that is displaced from the plane of motion of thesubstrate-holder support 419. The axes of motion of the printheads 412are parallel and displaced from each other laterally (horizontally). Theprintheads 412, in one embodiment, may be arranged linearly, or, asshown, in the form of a square or rectangle. The substrate-holdersupport 419 and the fluid-reservoir holder support 422 are independentlymobile, but move in a coordinated manner to effect the deposition offluid droplets upon the substrates 416. Position sensing (e.g. usingposition encoders) allows automated monitoring of the location of themobile elements of the microarrayer assembly 410 and computer controlinhibits any undesired contact between the components of the assembly410. As before, the fluid reservoirs and substrates are respectivelydisposed on the substrate-holder support 419 and the fluid-reservoirholder support 422 and are accurately positioned within a known anddefined plane and at a known location within the plane.

In the four-printing-head architecture, two printheads 412 a and 412 bare initially alternating in interlaced “dip-and-deposit” actions (oneis recharging its deposit elements 414 in the fluid-reservoir 420, whilethe other is depositing onto the substrate 416, and then vice versa),and the other two printheads 416 c and 416 d, are washed and then waitto be used. When a wash cycle is required for the first pair ofprintheads 412 a, 412 b (for instance at the conclusion of theirdepositing a set of fluid samples at all desired deposit locations onall substrates 416 on the substrate-holder 418), thefluid-reservoir-holder support 422 and substrate-holder support 419 aremoved beneath the other pair of printheads 412 c, 412 d which then takeover the printing operations. For each pair of printheads, in oneembodiment, the interlaced dip-and-deposit actions follow the principlesoutlined in the “concurrent wash” method previously described for thetwo printing-head assembly illustrated in FIG. 11.

In other embodiments, the use of:

-   -   a) a plurality of independently mobile printheads, each        constrained to move in parallel single axes, coupled with    -   b) the motion of a fluid-reservoir in any direction within a        plane perpendicular to the single axes when disposed beneath the        printheads, and    -   c) independent motion of a substrate in any direction within a        plane perpendicular to the single axes but displaced from the        plane of motion of the fluid-reservoir when disposed beneath the        printheads can be extended to any number of printheads to        improve droplet deposition rates. All such embodiments are        included within the scope of the invention.        v) Microarrayer Architectures with a Plurality of Deposition        Engines

Microarrayer components that include inserts that engage datums onanother piece of equipment, as previously described, permit suchcomponents to be loaded into a microarrayer assembly with precise,repeatable positioning. For example, when the substrates aretop-referenced in the substrate-holder, the top-surfaces of thesubstrates may be positioned in a known plane and absolute position withrespect to the printhead. Similarly, the use of fluid-reservoir holdersincorporating inserts that include reference surfaces that engage datumsdisposed on a fluid-reservoir holder support, as previously described,permits such fluid-reservoir holders to be loaded into a microarrayerassembly with precise, repeatable positioning of the fluid-reservoirswith respect to the printhead. The ability to repeatably and accuratelyload substrate-holders and fluid-reservoirs or fluid-reservoir holdersinto a microarrayer assembly enables a variety of other architectures tobe developed that increase throughput. For instance, in one embodiment,a multi-engine microarrayer assembly increases throughput by arranging aplurality of “deposition engines” to operate together in a cooperativemanner. A “deposition engine,” as the term is used herein, includes thefunctionality to a) deposit fluid droplets upon substrates mounted onsubstrate-holders, b) optionally load and unload fluid-reservoirs froman external conveyor and c) load and unload substrate-holders from anexternal conveyor. Concatenating a plurality of modular depositionengines enables scalability in the design of a microarrayer apparatus toachieve a desired level of throughput (i.e. depositions per hour).Autonomous operation of such a microarrayer assembly is enabled by:

i) automated supply and return of fluid-reservoirs (or offluid-reservoir holders with fluid-reservoirs thereon) in aspiratingsystems between a fluid-reservoir storage and the deposition engines,for instance, by the conveyor, andii) automated supply and return of substrate-holders between asubstrate-holder storage and the deposition engines, for instance by aconveyor.

In addition, the ability to replenish source-material fluid-reservoirsand substrates during the printing process by re-stocking thefluid-reservoir storage and the substrate-holder storage facilitatescontinuous printing operation with minimal or no cessation in printingoperations.

With reference to FIG. 13, one embodiment of a microarrayer assembly 510that includes a plurality of deposition engines 515 is illustrated. Theassembly utilizes non-aspirating ink jet devices. A non aspiratingink-jet deposition device is a device that is fed a continuous stream ofsample fluid, (for instance via a tube from a large reservoir 517)thereby eliminating the need to aspirate fluid from a fluid-reservoir,such as a microplate. Each deposition engine 515 shown in FIG. 13represents a deposition engine using one or more non-aspirating ink jetdevices. Depositions are performed inside the deposition engines usingsubstantially the same spotting techniques as earlier described, and aretherefore not repeated here.

With continued reference to FIG. 13, substrate-holders 518 are initiallyinstalled in the substrate-holder storage 570 a. To deposit fluiddroplets upon the substrates 516, a substrate-holder 518 with substrates516 thereon is removed from the substrate-holder storage 570 a andpassed, via a conveying system 523, to deposition engine 515 a, whichloads the substrate-holder 518 by placing it on a substrate-holdersupport 519 and commences deposition operations upon the substrates 516thereon. Once the fluid samples of the deposit engine 515 a have beendeposited on all the substrates 516 on the substrate-holder 518, thesubstrate-holder 518 is moved from the deposit engine 515 a to thedeposit engine 515 b, and a new substrate-holder 518 is loaded intodeposit engine 515 a. When both engines 515 a and 515 b have completeddeposition operations, the first substrate-holder 518 is passed todeposit engine 515 c and the second substrate-holder 518 is passed todeposit engine 515 b, and a new substrate-holder 518 is removed from thesubstrate-holder storage 570 a and installed into the deposit engine 515a. The sequence of passing substrate-holders 518 from engine to enginecontinues until all engines have deposited all fluid samples on allsubstrates 516 of all substrate-holders 518, and all thesubstrate-holders 518 have been installed in substrate-holder storage570 b. Two substrate-holder storages 570 a and 570 b are shown in FIG.13, however in some embodiments, only a single substrate-holder storage570 is used to dispense and receive substrate-holders 518.

With reference to FIG. 14, another embodiment of a multiple depositionengine microarrayer assembly 610 is illustrated. In this embodiment,which uses solid pins, pens, or aspirating ink jet devices within eachdeposit engine 615, two conveyor systems 623, 624 are used. The firstconveyor 623 transfers substrate-holders 618 and the second conveyor 624transfers fluid-reservoir holders 621 with fluid reservoirs 620 securedthereon or alternatively a fluid reservoir 620 that is not secured on aholder 621. In the illustrated embodiment, four deposition engines 615are shown; however, any other number of deposition engines 615 can beconcatenated to achieve a desired level of throughput.

Several methods exist for operating the assembly of FIG. 14. In oneembodiment, four substrate-holders 618 are removed from thesubstrate-holder storage 670 a and installed in the four engines 615 a,615 b, 615 c, 615 d via the robotic conveyor system 623. Next, the firstfluid-reservoir holder 621 holding a fluid-reservoir 620 is removed fromthe fluid-reservoir holder storage 683 a and installed, via the roboticconveyor 624, in the deposit engine 615 a. All fluid samples in thefirst fluid-reservoir 620 are deposited on all the substrates 516 on thefirst substrate-holder 518. The first fluid-reservoir holder 621 is thentransferred to the deposit engine 615 b, via the robotic conveyor 624,and a second fluid-reservoir holder 621 is installed, from thefluid-reservoir holder storage 683 a into the deposit engine 615 a. Nowboth the deposit engine 615 a and the deposit engine 615 b operate todeposit all fluid samples from the respective fluid-reservoirs 620 ontoall the substrates 616 on the respective substrate-holders 618. Afterthe completion of spotting operations, the first fluid-reservoir holder621 is transferred to the deposit engine 615 c, the secondfluid-reservoir holder 621 is transferred to the deposit engine 615 b,and a new third fluid-reservoir holder 621 is installed in the depositengine 615 a, via the robotic conveyor 624. The engines 615 a, 615 b,615 c operate to deposit all fluid samples from the respectivefluid-reservoirs 620 onto all the substrates 616 on the respectivesubstrate-holders 618. The same process is repeated to spot from afourth fluid-reservoir 620 and so on. As a further step, the firstfluid-reservoir holder 621 holding the first fluid-reservoir 620 istransferred into the fluid-reservoir holder storage 683 b as thefluid-reservoirs 620 continue to pass down the line of depositionengines 615. The sequence of passing the fluid-reservoirs holders 621from engine to engine continues until all the fluid-reservoir holders621 are transferred to the fluid-reservoir holder storage 683 b. At thispoint, the four substrate-holders 618 installed in the four engines 615have received all fluid samples on all of their respective substrates616. Therefore, the substrate-holders 619 are removed from the depositengines 615 and transferred to the substrate-holder storage 670 b, andfour fresh substrate-holders 618 are loaded into the deposit engines 615via the robotic conveyor 623. The process described above is thenrepeated, but with the fluid-reservoir holders 621 now moving fromfluid-reservoir holder storage 683 b to fluid-reservoir holder storage683 a.

In another embodiment, rather than passing fluid-reservoirs 621 betweendeposit engines 615 as just described, substrates 616 or substrateholders 619 are passed between the deposit engines 615, and the fluidreservoirs 620 or fluid reservoir holders 621 are initially loaded intothe engines 615.

Deposition operations of the arrayer engines in a multi-engine assemblysuch as that described can be performed synchronously or asynchronously.In synchronous operation, the printheads, substrate-holder supports, andfluid-reservoir holder supports in each deposition engine move in unisonto deposit fluid on the substrates. In asynchronous operation, thedeposition actions involving any motion of printheads, substrates ormicroplates within an engine are independently controlled. However, thedeposition engines remain coordinated with respect to starting andceasing deposition operations and transferring of fluid-reservoirholders and substrate-holders between engines and storage centers.Various other components may be shared between the engines, such as acomputer control system and operator interfaces, a cover, a heating,cooling, and humidification control system, air filters, vacuums, watersupplies, and pressure supplies.

The multi-deposit engine microarrayer assemblies described above mayinclude any of the features earlier described, for instance, one, two orfour independently mobile printheads.

In further embodiments, the above microarrayer assemblies 10, 210, 310,410, 510, 610 may include a variety of other features. For instance, inone embodiment, a sensor is included for sensing the presence or absenceof a substrate-holder within the receptacle (or bay) of asubstrate-holder storage, or within a microarrayer, or within adeposition engine of a multi-engine microarrayer. The sensor can be ofany type including, but not limited to, optical, capacitive, inductive,magnetic, infra-red, radio frequency or electro-magnetic. A sensor basedon the making or breaking of an electrical circuit may also be used.Similar sensors may be used for sensing the presence or absence offluid-reservoirs or fluid-reservoir holders in the microarrayerassemblies. In another embodiment, a sensor may be included for sensingthe height of fluid within each well of a fluid reservoir. The presenceof fluid heights outside a desired specified range may adversely effectthe fluid capture by the deposition element and the consistency of fluiddroplet deposition on the substrates. The sensor can be of any typeincluding, but not limited to, optical (direct measurement usingmodulated transmission, or indirect measurement using physicaldisplacement of a beam reflected from the fluid surface at an angleother than perpendicular incidence), and infra-red or radio frequency.

Another use for the microarrayers described above is to produce cellarrays. Cell arrays are composed of individual cells (or smallquantities of cells) deposited in ordered arrays upon a substrate suchas a glass slide or a multi-well target plate. Whereas the depositedvolume and the size of the printheads used to produce cell arrays may belarger than that used for genomic or proteomic microarrays, such arraysmay be generated by the techniques and methods disclosed herein and areincluded with the scope of the present invention.

vi) Spotting a Binding Agent on a Substrate:

Microarray substrates used for the capture of biological materials areusually coated over their entire surface with a material that bindsbiological molecules. Typically, a certain first set of biologicalmolecules are spotted onto the coating in specific spot locations tobind to the coating in those locations. A sample of biological materialunder test is then effectively washed over the set of spots such thatbiological interactions between the set of molecules first spotted andthe sample can be identified by locating the attachment of the sample tothe substrate. It is common, however, in genomic and proteomicmicroarray experiments for the sample to also bind to the slide coatingin an undesired, non-specific manner (i.e. not related to a particulargenome sequence or fold structure).

A potential solution for this problem is to only place the “bindingagent” material on the slide in the location where the first set ofbiological molecules are to be placed. The rest of the substrate'ssurface can be left bare, or with a coating of material that willinhibit or suppress non-specific binding. Using this concept, thebinding agent can be deposited on the substrate in spot locations thatare later re-spotted with the set of biological molecules. Some of themicroarrayer embodiments herein described, in particular a) the abilityto accurately position substrates repeatedly under a printhead using asubstrate-holder that is positioned in a known location in a plane andb) the provision and use of multiple printheads, may be used to realizethe above technique. Using the architectures described, a series ofmaterials having different functions, may be spotted onto the samelocation on a substrate. It is also possible to deposit the sample onlyat the locations of the first set of biological molecules, instead ofwashing it over the entire array, resulting in significant reduction inthe amount of sample required

2) Tissue Arrayer Embodiments

The embodiments described in the foregoing for the dispensing of fluiddroplets in the form of microarrays are readily adapted to an apparatusfor the deposition of semi-solid or solid tissue samples in orderedarrays.

With reference to FIG. 15, in one embodiment of a tissue arrayer 710,donor and receiver blocks 716, 717, for example, made of paraffin, aremounted in a block-holders 718 that include top-referencing to maintainthe top of the blocks 716, 717 in a known, consistent plane. The systemis similar to that described in FIG. 3 for use with substrates. The topsurface of the paraffin blocks are pressed from below against areference surfaces 740 (or reference elements), of the block-holders 718that are machined and/or constructed to ensure that the top surface 725of each block 716, 717 is in a desired plane, and that the block's topsurface 725 is substantially coplanar with that of every other block716, 717 held by the block-holder 718, if more than one block 716, 717is installed in the holder 718. The block 716, 717 is held against theblock-holder's reference surfaces 740 by means of spring clips or otherlocking mechanisms with resilient or biasing members. In anotherembodiment, a removable block-mounting fixture similar to that describedin FIGS. 4A-4C for use with substrates is provided for use with donorand receiver blocks 716, 717. One advantage of using top-referencing asdescribed is that the donor and receiver blocks 716, 717 are repeatablylocated in the block-holder 718 at each loading. This facilitatesconsistent and accurate removal of tissue cores from the donor-block 716and consistent and accurate placement of tissue cores in the receiverblock 717.

In another embodiment, the block-holders 718 include inserts designed torest upon datums disposed on a mobile block-holder support 789. Thereferencing systems previously described for use with the microarrayersubstrate holder, the substrate-holder support, the fluid-reservoirholder, and the fluid-reservoir holder are also applicable to theblock-holder 718 and the block-holder support 789 and will therefore notbe further described. The referencing system permits installation of theblock-holders 718 onto the mobile block-holder supports 789 of thetissue arrayer 710 in a predictable, repeatable manner such that the topsurfaces 725 of the donor and receiver blocks 716, 717 may be locatedaccurately within a known plane with respect to a coring head 750.

In one embodiment, overall block-holder and block-holder support systemposition accuracy is within ±0.02″ in the x, y, and z-axes. In apreferred embodiment, overall block-holder and block-holder supportsystem position accuracy is within ±0.002″ in the z-axis and within±0.01″ in the x and y axes. In a more preferred embodiment, overallblock-holder and block-holder support system position accuracy is within±0.0002″ in the z-axis and within ±0.001″ in the x and y axes.

Similar to the microarrayer architectures described earlier, the use ofblock holders 718 and block-holder supports 789 that include areferencing system as described may be beneficially combined withblock-holder storages 770 a, 770 b for temporally storing a multiplicityof block-holders 718 and the blocks 716, 717 thereon (either donorblocks or recipient blocks). In another embodiment, a conveyor systemfor removing and delivering block-holders 718 from and to theblock-holder storages 770 a, 770 b may be included. In theseembodiments, since manual loading/unloading of blocks 716, 717 from thesection of the arrayer dedicated to coring/core-deposition iseliminated, many sources of error resulting from frequent human accessto the coring area are minimized or eliminated.

Another benefit of the present invention is that block-holders 718 arereadily loaded into, and removed from, the coring/deposition area of thetissue arrayer 710 without loss of positional accuracy (i.e. allproperly prepared block-holders 718, when mounted on the block-holdersupport 719, will have their top surfaces 725 in the same plane, and atthe same location and orientation.)

Another benefit produced by various embodiments of the invention is thatthe number of blocks 716, 717 that can be processed by the tissuearrayer 710 is limited only by the number of blocks 716, 717 on eachblock-holder 718 and the available number of block-holders 718 in theblock-holder storages 770 a, 770 b. Moreover, in one embodiment,block-holders 718 can be removed from, and added to, the block-holderstorages 770 a, 770 b while coring/core-depositions are underway on anactive block-holder 718 loaded in the coring/deposition area of thearrayer 710. This facilitates continuous operation of the tissue arrayer710.

Another benefit derived from various embodiments of the invention isthat relatively small block-holders 718, holding, for example, 2 to 10paraffin blocks 716, 717 can be used, since the number of blocks 716,717 that can be processed is limited only by the capacity of theblock-holder storage 770 and not the size of the block-holder 718. Theuse of smaller block-holders 718 enables the size of the deposition areaand the volume of the tissue arrayer 710 to be reduced.

In another embodiment, scalable tissue arrayer designs are possible,since the size and functions of the coring/core-deposition equipment isno longer tied to the number of blocks 716, 717 that can be processed.For instance, embodiments using multi-engine tissue arrayers, using theprinciples discussed earlier for multi-engine microarrayers, can bedeveloped.

With reference to FIG. 15, an embodiment of an automated tissue arrayer710 is illustrated. Both the donor blocks 716 and the receiver blocks717 may be moved in any direction within a horizontal X-Y plane whendisposed beneath the coring head 750. The coring head 750 is constrainedto move in an axis perpendicular to the X-Y plane. The coring head 750includes two coring needles 752, 754. The first needle 752 coresreceiver volumes in the receiver-blocks 717, and the second needle 754extracts the tissue cores from the donor block 716 and deposits them inthe aforementioned receiver volumes in the receiver block 717. In oneembodiment, paraffin cores removed from the receiver block 717 aredeposited into the voids left after coring of the donor block 716 tohelp maintain the latter's structural integrity. In another embodiment,liquid or semi-solid paraffin, or other suitable material, is injectedinto the voids of the donor-block 716 to maintain the latter'sstructural integrity. In a further embodiment, a needle 756 fordispensing liquid or semi-solid paraffin is vertically mobile and ismounted on a second vertical axis 751, offset laterally from the coringhead 750 that holds first and second needles 752, 754.

The first and second needles 752, 754 are mounted to a linear verticalstage that is controlled by a computer 760. The vertical stage hasencoders (linear or rotary) to provide positional feedback. The positionof the needles 752, 754 in the vertical axis for coring and depositionoperations is determined by the computer 760 under closed loop control.The donor blocks 716 are top referenced and are mounted in block-holders718. The donor block-holder 718 rests upon datums 764 disposed on ashared platen 789, as previously described. Similarly, thereceiver-blocks 717 are top referenced and are mounted in theblock-holders 718. In one embodiment, a conveyor moves donorblock-holders 718 to and from a donor block-holder storage 770 a.Similarly, in another embodiment, a conveyor moves receiver-blockholders to and from a donor-block holder storage 770 b.

In other embodiments in accordance with the invention, in the same waythat separate, parallel planes of motion were used for thesubstrate-holder and the fluid-reservoir holder in the motionarchitectures disclosed for microarrayers, the donor and receiver-blockholders 718 may also be so arranged with separate independent planes ofmotion. Moreover, the planes of motion of the donor and receiver blockholders 718, when under the coring head 750, may also be perpendicularto the axis of motion of the coring head 750.

With continued reference to FIG. 15, in another tissue arrayerembodiment, a high resolution camera system 780 and a high resolutionvideo display 782 are also included. In one embodiment the camera system780 mounted within the tissue arrayer 710 and the high resolutiondisplay 782 are located in different locations, such that remoteexamination and targeting of core locations in the tissue sample ispossible. The camera system 780 and the coring head 750 are securelymounted on the same fixed bridge such that a known fixed offset in X andY dimensions exists between them. In one embodiment, a known referencemark or series of reference marks are provided within the camerasystem's 780 field of view to establish the distance of separationbetween the coring head 750 and the camera 780 and/or to correct fornon-linearities in the video image.

As an example, the tissue arrayer 710 may be used as follows. As a firststep the donor-block 716, mounted in a known plane on a donor blockholder 718 is moved in the X-Y plane under the high-resolution camerasystem 780, which is mounted to provide an image of the top surface 725of the donor-block 716. A high resolution image of the donor block'stissue sample is displayed on the high resolution monitor 782. In thenext step, using an animated pointing device such as a computer mouse,an operator moves a pointer, such as a computer cursor on the highresolution monitor 782, over the image of the tissue sample, anddesignates locations on the sample from which tissue cores are to betaken. In some embodiments, desired coring locations on a multiplicityof donor-blocks 716 are specified, by removing them, in turn, from adonor-block holder storage 770 a, defining coring locations, andreturning them to the storage 770 a. A computer system 760 is used tostore the X-Y coordinates of the desired coring locations. In the nextstep, an operator initiates automatic coring operations, such that thedonor blocks 716 are transported between the donor-block holder storage770 a and the coring area, and receiver blocks 717 are transportedbetween the receiver block holder storage 770 b and the coring area,until all coring and deposition actions are completed.

3) Fluidics Robots

Many of the inventions herein disclosed for dispensing fluid droplets inthe form of microarrays are also directly applicable to the fluiddispensing requirements of fluidics robots. Fluid dispensingapplications handled by fluidic robots may include, for example:

-   -   a) dispensing fluid from external reservoirs into arrays of        smaller receptacles, such as micro-centrifuge tubes or        microplates;    -   b) transferring fluid from one fluid reservoir mounted on the        robot's platen to another reservoir, e.g. dispensing the        contents of a centrifuge tube into the wells of a microplate;    -   c) transferring fluid from the wells of one microplate to the        same or different wells of another microplate;    -   d) dividing fluid samples from one microplate into multiple        microplates with the same distribution, (such action is commonly        known as “replication” of microplates);    -   e) dividing fluid samples from one microplate into multiple        microplates with a different distribution;    -   f)transferring fluids from microplates with a less dense        arrangement of wells to microplates with a denser arrangement of        wells (e.g. transfers from four 96-well microplates to one        384-well microplates, or from four 384-well microplates to one        1536-well microplate) (such actions are commonly known as        “compression” of microplates);    -   g) transferring fluids from microplates with a denser        arrangement of wells to microplates with a less dense        arrangement of wells (e.g. transfers from one 1536-well        microplate to four 384-well microplates, or from one 384-well        microplate to four 96-well microplates) (such actions are        commonly known as “expansion” of microplates);    -   h) transferring fluids from particular wells of one or more        microplates to a new microplate (such actions are commonly known        as “re-arraying” or “cherry picking”);    -   i) compressing plates, where, for example, the contents of four        96-well microplates are combined onto one 384-well microplate;        and    -   j) preparing assays, wherein several fluids are dispensed into a        vessel (such as the well of a microplate) for the purpose of        causing a chemical or biological reaction.

With reference to FIG. 16, one embodiment of a fluidic robot 810 isillustrated. The robot 810 includes a dispensing head 812 that is mobilein a vertical axis. In one embodiment, the dispensing head 812 includesdispensing tubes 890 connected to external fluid reservoirs (not shown).In another embodiment, the dispensing head 812 includes pipette tips 891for aspirating and dispensing fluid from local source reservoirs 816,817. The dispensing tubes and the pipette tips 891 may be moveablerelative to each other in at least one of an x-axis or y-axis toaccommodate a variety of well separations (i.e. the center-to-centerdistance between wells) on source and target reservoirs 816, 817. In oneembodiment, a vacuum source 892 is provided to aspirate fluids from thefluid reservoirs. Similarly, a pressure source may be provided to ejectfluid from the dispensing head 812.

The robot also includes a source-reservoir 816 mounted to asource-reservoir holder 818, the source-reservoir holder 818 includinginserts for resting on datums 864 disposed on a source-reservoir holdersupport 819. The source-reservoir holder 818 may move in any directionwithin a plane perpendicular to the axis of motion of the dispensinghead 812 when disposed beneath the dispensing head 812. A conveyor 823may be used to extract source-reservoir holders 816 from asource-reservoir holder storage 870 and to return them thereto.

Similarly, the robot 810 also includes a target-reservoir 817 mounted toa target-reservoir holder 821, the target reservoir holder 821 includinginserts for resting on datums 864 disposed on a target reservoir holdersupport 822. The target-reservoir holder 821 may move in any directionwithin a plane perpendicular to the axis of motion of the dispensinghead 821 and displaced from, but parallel to, the plane of motion of thesource reservoir holder 819. A conveyor 825 may be used to extracttarget-reservoir holders 819 from a target-reservoir holder 883 andreturn them thereto.

The referencing systems previously described for use with themicroarrayer substrate holder, the substrate-holder support, thefluid-reservoir holder, and the fluid-reservoir holder support are alsoapplicable to the source-reservoir holder 818, the source-reservoirholder support 819, the target-reservoir holder 821, and thetarget-reservoir holder support 822 and will therefore not be furtherdescribed. The referencing system permits installation of the holders818, 821 onto the mobile reservoir holder supports 819, 822 of thefluidics robot 810 in a predictable, repeatable manner.

In one embodiment, overall holder and holder support system positionaccuracy is within ±0.02″ in the x, y, and z-axes. In a preferredembodiment overall holder and holder support system position accuracy iswithin ±0.002″ in the z-axis and within ±0.01″ in the x and y axes. In amore preferred embodiment, overall holder and holder support systemposition accuracy is within ±0.0002″ in the z-axis and within ±0.001″ inthe x and y axes.

The use of standardized holders that may be repeatably and accuratelyloaded into the fluidics robot 810 in a known position within a knownplane, reduces the need to manually reconfigure the dispensingassemblies of the robot 810 for different operating conditions. As acorollary, the need for human access to the dispensing area of themachine is minimized, reducing the potential for human error. Moreover,the system facilitates the conversion of the robot 810 from one transferoperation to another. For instance, to change the transfer operationbeing conducted by the robot 810, a user places the new source reservoir816 and target reservoir 817 in the storages 870, 883, and updates thecomputer control system 893 of the fluidic robot 810 to inform thecomputer 893 of the type of fluid reservoirs 816, 817 being held in thestorages 870, 883, their locations, and the type of transfer operationto be conducted.

In another embodiment in accordance with the invention, to avoidcross-contamination between fluid samples, disposable pipette tips 891are used (which are then discarded after pipetting of that sample iscompleted). Alternatively, in another embodiment, a wash station 824 isprovided to wash the pipette tips 891 before a new fluid sample isaspirated. In many instances, both a washing station 824 and disposablepipette tips 891 are provided. With continued reference to FIG. 16, apipette tip replacement assembly is illustrated. The assembly includes amobile pipette-tip support 895 that holds disposable pipette tips 891that may be supplied to the dispensing head 812. The pipette-tip support895 may also hold a bin for discarded pipette tips 891. The mobilepipette-tip holder 895 can move in any direction within a plane parallelto those of the source reservoir holder 818 and the target reservoirholder 821, but is vertically displaced from both. In a furtherembodiment, a pipette-tip-tray storage is included, from which aplurality of pipette-tip trays 895 may be retrieved that containing awide range of tip sizes and types.

In another embodiment, a plurality of dispensing heads are included inthe fluidics robot. Each dispensing head is independently mobile and isrestricted to travel in a vertical axis, the axes being spaced apart.The apparatus may be used, for example, in situations where a wide rangeof pipette tip sizes are required for transfer operations. In thissituation, each dispensing head may accommodate dispensers that accept adifferent range of pipette-tip sizes. In another example, each of thedispensing heads may accommodate a different range of well-to-wellspacings. As another example, one head of the assembly may be designedfor colony picking, such that colonies of cells grown on a growth mediain a container can be extracted and dispensed into other reservoirs,such as the well of a microplate.

In other embodiments, devices that further process the assays that havebeen prepared may be added to the system 810. For example, the sourcereservoir 816 or the target reservoir 817 may be transferred to one ormore various devices, including, but not limited to:

-   -   a) a device for supporting polymerase chain reactions via timed        heating and cooling actions,    -   b) a device for maintaining timed exposure to a specific        temperature and/or humidity (for instance for a hybridization        reaction, a re-hydration step or a cooling step to slow a        reaction),    -   c) a device for timed heating and cooling at other than ambient        air pressure,    -   d) a device for centrifugation of the fluid samples,    -   e) a device for vacuum filtering of fluids (including vacuum        filtering in a well-plate format),    -   f) a device for filtering of plasmids by magnetic beads        (including magnetic bead filtering in a well-plate format),    -   g) a device for shaking and stirring,    -   h) a device for producing optical images of the dispensed or        deposited material,    -   i) a device for detecting the presence of substances based on        adsorption, or    -   j) a scanner for producing images of concentrations of tags        attached to biological entities, such tags being detectable due        to radio-active emission, florescent emission following laser        illumination, or optical scattering (such as that for minute        optical scatterers known as quantum dots).

In one embodiment, fluid dispensing operation can continue in thedispensing area of the assembly while other holders 818, 821 are beingprocessed in the additional devices.

4) Identification and Tracking Adaptations

In other embodiments of the foregoing microarrayer, tissue arrayer andfluid dispensing systems, the use of one or more of the followingelements may be included to improve their overall performance orutility. These elements also can be beneficially used in otherinstruments for the generation, dispensing, processing, sampling,scanning and examination of deposited fluid, semi-solid or solidsamples.

For instance, in various embodiments, a means of identification may beprovided on one or more of the apparatuses that are loaded into orremoved from, any of the assemblies earlier described. As an example,some of the elements that may effectively receive such identificationmeans include:

-   -   a) the microarray substrates, such as glass slides,    -   b) the microarray substrate-holders,    -   c) the microarray fluid reservoirs, such as microplates,    -   d) the microarray fluid-reservoir holders,    -   e) the tissue arrayer donor blocks,    -   f) the tissue arrayer donor-block holders,    -   g) the tissue arrayer receiver blocks,    -   h) the tissue arrayer receiver-block holders,    -   i) the fluidics robot source-reservoirs, such as microplates,    -   j) the fluidics robot source-reservoir holders,    -   k) the fluidics robot target-reservoirs,    -   l) the fluidics robot target-reservoir holders, and    -   m) the fluidic robot pipette-tip holders.

The identification allows the element to be recognized and/or itsprogress tracked and correlated with information recorded elsewhere onthe processes that have been applied to that element. For example,tracking a unique identification code on a microarray slide permits thatslide to be located within a collection of slides. If the microarrayercontrol computer records the details of the fluid samples deposited onthat slide, and where they are deposited, both the slide and theinformation on its data contents is easily retrieved. As anotherexample, pipette-tip holders of different types and sizes of may beautomatically recognized and appropriately chosen by the fluidic robot.Similarly, the fluidics robot may autonomously recognize the type offluid reservoirs placed in the various receptacle of the storage hoteland retrieve them accordingly. Several means exist to provideidentification of these elements including, but not limited to:

-   -   I. placement, on the element, of a bar code that can be        optically scanned by non-contact means,    -   II. placement, on the element, of a radio-frequency        identification (RFID) transponder that is programmed with a        unique code, that can be read by a RFID interrogator by        non-contact means,    -   III. placement, on the element, of a semi-conductor memory        device that is programmed with a unique code, that can be read        by a electrical sensor by direct electrical contact, and    -   IV. placement, on the element, of a semi-conductor memory device        that is programmed with a unique code, that can be read over an        optical, infra-red or radio-frequency communication to an        external sensor.

In other embodiments, a means of storing identification, content andprocess data is provided on one or more of those elements that areloaded into, or removed from, any of the assemblies described herein.The local storage on an element of both identification and contentinformation may provide various advantages. For example, the contents ofa microplate retrieved from a stack of similar plates otherwiseidentical in appearance can be unambiguously identified. As anotherexample, a microarrayer can sense and internally record the informationon the content of each well location from which fluid is sampled; thisinformation can be transferred, by the microarrayer, to the local datarecording associated with the substrate onto which the material isdeposited (or the substrate-holder). As another example, a fluidicsrobot preparing an assay in the well of a target reservoir can sense thecontents of all contributing source fluid reservoirs and record allinformation on the local data storage of the target reservoir. In yetanother example with the fluidics robot, the polymerase chain reaction(PCR) protocol applied to particular fluid samples in a microplate maybe recorded in the local data storage of the reservoir and passed alongwith the sample in all subsequent processing. Many similar uses for thelocal data storage exist and are included within the scope of thepresent invention. Several means exist to provide identification ofthese elements including, but not limited to:

-   -   I. placement, on the element, of a radio-frequency        identification (RFID) transponder that is dynamically        programmable with information on the element, that can be read        by a RFID interrogator by non-contact means,    -   II. placement, on the element, of a semi-conductor memory device        that is dynamically programmable with information about the        element, that can be read by a electrical sensor by direct        electrical contact, and    -   III. placement, on the element, of a semi-conductor memory        device that is dynamically programmable with information about        the element, that can be read over an optical, infra-red or        radio-frequency communication to an external sensor.

Other embodiments incorporating the concepts disclosed herein may beused without departing from the spirit and scope of the invention. Thedescribed embodiments are to be considered in all respects as onlyillustrative and not restrictive.

What is claimed is: 1.-108. (canceled)
 109. A method of depositingminute droplets of fluid on a substrate comprising: operativelyconnecting a plurality of deposition engines, each deposition enginecomprising at least one printhead comprising at least one depositelement adapted to deposit minute droplets of fluid onto a surface ofthe substrate; and transferring at least one holder between thedeposition engines, the holder: adapted to hold at least one of asubstrate and a fluid reservoir and comprising an apparatus forprecisely and repeatably positioning the holder on a support.
 110. Themethod of claim 109, wherein each deposition engine comprises aplurality of printheads.
 111. The method of claim 109, furthercomprising the step of transferring at least one holder from a firsthotel to a first deposition engine.
 112. The method of claim 111,further comprising the step of transferring at least one holder from thefirst deposition engine to a second hotel.
 113. (canceled)
 114. Amicroarray produced in accordance with the method of claim 109.115.-119. (canceled)
 120. The method of claim 112, wherein at least oneof (i) the step of transferring the holder from the first hotel to thefirst deposition engine and (ii) the step of transferring the holderfrom the first deposition engine to the second hotel, occurs while atleast one other deposition engine is performing at least one of a fluidcapture operation and a droplet deposition operation.
 121. The method ofclaim 112, further comprising: re-stocking the first hotel with at leastone additional holder while at least one deposition engine is performingat least one of a fluid capture and a droplet deposition operation. 122.The method of claim 121, wherein the first hotel and the second hotelcomprise a same hotel.
 123. The method of claim 109, wherein the holdercomprises at least one holder adapted to hold the substrate and at leastone holder adapted to hold the fluid reservoir.
 124. The method of claim123, wherein (i) the holder adapted to hold the substrate is transferredbetween the deposition engines and (ii) the holder adapted to hold thefluid reservoir is installed in a single deposition engine.
 125. Themethod of claim 109, wherein the apparatus for precisely and repeatablypositioning the holder on a support comprises a datum plane defined byat least three datums.
 126. The method of claim 109, wherein the depositelement comprises a solid pin.
 127. A microarrayer assembly comprising:a plurality of operatively connected deposition engines, wherein eachdeposition engine comprises at least one printhead comprising at leastone deposit element adapted to deposit minute droplets of fluid onto asurface of a substrate; and an automated supply and return apparatusadapted to transfer at least one holder between the deposition engines,the holder: adapted to hold at least one of a substrate and a fluidreservoir, and comprising an apparatus for precisely and repeatablypositioning the holder on a support.
 128. The microarrayer of claim 127,wherein at least one deposition engine comprises a plurality ofprintheads.
 129. The microarrayer of claim 127, wherein at least onedeposition engine comprises a wash station adapted to wash the depositelement.
 130. The microarrayer of claim 129, wherein the plurality ofdeposition engines operate synchronously with each other.
 131. Themicroarrayer of claim 127, wherein the plurality of deposition enginesoperate asynchronously with each other.
 132. The microarrayer of claim127, wherein the deposit element comprises a solid pin.
 133. Themicroarrayer of claim 127, wherein the automated supply and returnapparatus comprises a conveyor system.
 134. The microarrayer of claim127, wherein the holder comprises at least one holder adapted to holdthe substrate and at least one holder adapted to hold the fluidreservoir.
 135. The microarrayer of claim 134, wherein the automatedsupply and return apparatus comprises: a first apparatus fortransferring the holder adapted to hold the substrate, and a secondapparatus for transferring the holder adapted to hold the fluidreservoir.
 136. The microarrayer of claim 134, wherein (i) the holderadapted to hold the substrate is adapted to be transferred between thedeposition engines and (ii) the holder adapted to hold the fluidreservoir is adapted to be installed in a single deposition engine. 137.The microarrayer of claim 127, further comprising: at least one hoteladapted to store at least one holder, the hotel operatively connected tothe automated supply and return apparatus.
 138. The microarrayer ofclaim 137, wherein the automated supply and return apparatus is adaptedto transfer the holder from a first hotel to a first deposition engine.139. The microarrayer of claim 138, wherein the automated supply andreturn apparatus is further adapted to transfer the holder from thefirst deposition engine to a second hotel.
 140. The microarrayer ofclaim 139, wherein the automated supply and return apparatus is adaptedto at least one of (i) transfer the holder from the first hotel to thefirst deposition engine and (ii) transfer the holder from the firstdeposition engine to the second hotel, while at least one otherdeposition engine is performing at least one of a fluid captureoperation and a droplet deposition operation.
 141. The microarrayer ofclaim 138, wherein the first hotel is adapted to be re-stocked while atleast one deposition engine is performing at least one of a fluidcapture operation and a droplet deposition operation.
 142. Themicroarrayer of claim 141, wherein the first hotel and the second hotelcomprise a same hotel.
 143. The microarrayer of claim 127, furthercomprising at least two hotels, wherein one hotel is adapted to store atleast one substrate holder and another hotel is adapted to store atleast one fluid reservoir holder.
 144. The microarrayer of claim 127,further comprising a controller adapted to coordinate the operations ofthe deposition engines and the automated supply and return apparatus.